Compositions for manipulating the lifespan and stress response of cells and organisms

ABSTRACT

Provided herein are methods and compositions for modulating the activity of sirtuin deacetylase protein family members; p53 activity; apoptosis; lifespan and sensitivity to stress of cells and organisms. Exemplary methods comprise contacting a cell with an activating compound, such as a flavone, stilbene, flavanone, isoflavone, catechin, chalcone, tannin or anthocyanidin; or an inhibitory compound, such as a sphingolipid, e.g., sphingosine.

This patent application claims the benefit of priority from U.S.Provisional Patent Application No. 60/532,158, filed Dec. 23, 2003 andU.S. Provisional Patent Application No. 60/483,949, filed Jul. 1, 2003,each of which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

There is now good evidence from model organisms that the pace of agingcan be regulated (Kenyon, C. Cell 105, 165-168 (2001)). Longevityregulatory genes have been identified in many eukaryotes, includingrodents, flies, nematode worms and even single-celled organisms such asbaker's yeast (reviewed in Sinclair, D. Mech Ageing Dev 123, 857-67(2002); Hekimi, S. & Guarente, L. Genetics and the specificity of theaging process. Science 299, 1351-4 (2003)). These genes appear to bepart of an evolutionarily conserved longevity pathway that evolved topromote survival in response to deteriorating environmental conditions(Kenyon, C. Cell 105, 165-168 (2001); Guarente, L. and Kenyon, C. Nature408, 255-62. (2000). The yeast S. cerevisiae has proven a particularlyuseful model in which to study cell autonomous pathways of longevity(Sinclair, D. Mech Ageing Dev 123, 857-67 (2002)). In this organism,replicative lifespan is defined as the number of daughter cells anindividual mother cell produces before dying. Yeast lifespan extensionis governed by PNC1, a calorie restriction (CR)—and stress-responsivegene that depletes nicotinamide, a potent inhibitor of the longevityprotein Sir2. Both PNC1 and SIR2 are required for lifespan extension byCR or mild stress (Lin et al. Science 289, 2126-8 (2000); Anderson etal. Nature 423, 181-5 (2003)) and additional copies of these genesextend lifespan 30-70% (Lin et al. Science 289, 2126-8 (2000); Andersonet al. Nature 423, 181-5 (2003); Kaeberlein et al. Genes Dev 13, 2570-80(1999). Based on these results we proposed that CR may confer healthbenefits in a variety of species because it is a mild stress thatinduces a sirtuin-mediated organismal defense response (Anderson et al.Nature 423, 181-5 (2003).

Sir2, a histone deacetylase (HDAC), is the founding member of thesirtuin deacetylase family, which is characterized by a requirement forNAD⁺ as a co-substrate (Landry et al. Proc Natl Acad Sci USA 97, 5807-11(2000); Imai et al. Nature 403, 795-800 (2000); Smith et al. Proc NatlAcad Sci USA 97, 6658-63 (2000); Tanner et al. Proc Natl Acad Sci USA97, 14178-82 (2000); Tanny et al. Cell 99, 735-45 (1999); Tanny, J. C.and Moazed, D. Proc Natl Acad Sci USA 98, 415-20 (2001)). SIR2 wasoriginally identified as a gene required for the formation oftranscriptionally silent heterochromatin at yeast mating-type loci(Laurenson, P. and Rine, J. Microbiol Rev 56, 543-60. (1992)).Subsequent studies have shown that Sir2 suppresses recombination betweenrepetitive DNA sequences at ribosomal RNA genes (rDNA)(Smith, J. S. andBoeke, J. D. Genes Dev 11, 241-54 (1997); Bryk et al. Genes Dev 11,255-69 (1997); Gottlieb, S. and Esposito, R. E. Cell 56, 771-6 (1989)).Sir2 has also been implicated in the partitioning of carbonylatedproteins to yeast mother cells during budding (Aguilaniu et al. Science(2003). Studies in C. elegans, mammalian cells, and the single-celledparasite Leishmania, indicate that the survival and longevity functionsof sirtuins are conserved (Tissenbaum, H. A. and Guarente, L. Nature410, 227-30 (2001); Vaziri et al. Cell 107, 149-59 (2001); Luo et al.Cell 107, 137-48 (2001); Vergnes et al. Gene 296, 139-50 (2002)). In C.elegans additional copies of sir-2.1 extend lifespan by 50% via theinsulin/IGF-1 signalling pathway, the same pathway recently shown toregulate lifespan in rodents (Holzenberger et al. Nature 421, 182-7(2003); Shimokawa et al. Faseb J 17, 1108-9 (2003); Tatar et al. Science299, 1346-51 (2003)).

SUMMARY OF THE INVENTION

Provided herein are methods for activating a sirtuin deacetylase proteinfamily member. The method may comprise contacting a sirtuin deacetylaseprotein family member with a compound having a structure selected fromthe group of formulas 1-25 and 31. Compounds falling within formulas1-25 and 31 and activating a sirtuin protein are referred to herein as“activating compounds.” The activating compound may be a polyphenolcompound, such as a plant polyphenol or an analog or derivative thereof.Exemplary compounds are selected from the group consisting of flavones,stilbenes, flavanones, isoflavones, catechins, chalcones, tannins andanthocyanidins or analog or derivative thereof. In illustrativeembodiments, compounds are selected from the group of resveratrol,butein, piceatannol, isoliquiritgenin, fisetin, luteolin,3,6,3′,4′-tetrahydroxyflavone, quercetin, and analogs and derivativesthereof. In certain embodiments, if the activating compound is anaturally occurring compound, it may not in a form in which it isnaturally occurring.

The sirtuin deacetylase protein family member maybe the human SIRT1protein or the yeast Sir2 protein.

The sirtuin deacetylase protein family member may be in a cell, in whichcase the method may comprise contacting the cell with an activatingcompound or introducing a compound into the cell. The cell may be invitro. The cell may be a cell of a subject. The cell may be in a subjectand the method may comprise administering the activating compound to thesubject. Methods may further comprise determining the activity of thesirtuin deacetylase protein family member.

A cell may be contacted with an activating compound at a concentrationof 0.1-100 μM. In certain embodiments, a cell is further contacted withan additional activating compound. In other embodiments, a cell iscontacted with a least three different activating compounds.

Other methods encompassed herein include methods for inhibiting theactivity of p53 in a cell and optionally protecting the cell againstapoptosis, e.g., comprising contacting the cell with an activatingcompound at a concentration of less than about 0.5 μM. Another methodcomprises stimulating the activity of p53 in a cell and optionallyinducing apoptosis in the cell, comprising contacting the cell with anactivating compound at a concentration of at least 50 μM.

Also provided herein is a method for extending the lifespan of aeukaryotic cell, such as by increasing its resistance to stress,comprising contacting the cell with a compound selected from the groupconsisting of stilbene, flavone and chalcone family members. Suchcompounds are referred to as “lifespan extending compounds.” Thecompound may have the structure set forth in formula 7. Other compoundsmay be activating compounds having a structure set forth in any offormulas 1-25 and 30, provided they extend lifespan or increaseresistance to stress. The compound may be selected from the groupconsisting of resveratrol, butein and fisetin and analogs andderivatives thereof. In certain embodiments, if the lifespan extendingcompound is a naturally occurring compound, it is not in a form in whichit is naturally occurring. The method may further comprise determiningthe lifespan of the cell. The method may also further comprisecontacting the cell with an additional compound or with at least threecompounds selected from the group consisting of stilbene, flavone andchalcone family members or other lifespan extending compound. The cellmay be contacted with a compound at a concentration of less than about10 μM or at a concentration of about 10-100 μM. The cell may be in vitroor in vivo, it may be a yeast cell or a mammalian cell. If the cell isin a subject, the method may comprise administering the compound to thesubject.

Methods for inhibiting sirtuins; inhibiting deacetylation of p53;stimulating apoptosis; shorting lifespan and rendering cells andorganisms sensitive to stress are also encompassed. One method comprisescontacting a sirtuin or cell or organism comprising such with aninhibitory compound having a formula selected from the group of formulas26-29 and 31.

Also provided herein are compositions comprising, e.g., two compoundseach having a formula selected from the group of formulas 1-31. Furtherprovided herein are screening methods for identifying compounds, e.g.,small molecules, that modulate sirtuins and/or modulate the life span orresistance to stress of cells. Methods may comprise (i) contacting acell comprising a SIRT1 protein with a peptide of p53 comprising anacetylated residue 382 in the presence of an inhibitor of class I andclass II HDAC under conditions appropriate for SIRT1 to deacetylate thepeptide and (ii) determining the level of acetylation of the peptide,wherein a different level of acetylation of the peptide in the presenceof the test compound relative to the absence of the test compoundindicates that the test compound modulates SIRT1 in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a through 1 d show the effects of resveratrol on the kinetics ofrecombinant human SIRT1. FIG. 1 a shows resveratrol dose-response ofSIRT1 catalytic rate at 25 μM NAD⁺, 25 μM p53-382 acetylated peptide.Relative initial rates are the mean of two determinations, each derivedfrom the slopes of fluorescence (arbitrary fluorescence units, AFU) vs.time plots with data obtained at 0, 5, 10 and 20 min. of deacetylation.FIG. 1 b shows the SIRT1 initial rate at 3 mM NAD⁺, as a function ofp53-382 acetylated peptide concentration in the presence (Δ) or absence(▪) of 100 μM resveratrol. Lines represent non-linear least-squares fitsto the Michaelis-Menten equation. Kinetic constants: K_(m)(control,▪)=64 μM, K_(m)(+resveratrol, Δ)=1.8 μM; V_(max)(control, ▪)=1107AFU/min., V_(max)(+resveratrol, Δ)=926 AFU/min. FIG. 1 c shows the SIRT1initial rate at 1 mM p53-382 acetylated peptide, as a function of NAD⁺concentration, in the presence (Δ) or absence (▪) of 100 μM resveratrol.Lines represent non-linear least-squares fits to the Michaelis-Mentenequation. Kinetic constants: K_(m)(control, ▪)=558 μM,K_(m)(+resveratrol, Δ)=101 μM; V_(max)(control, ▪)=1863 AFU/min.,V_(max)(+resveratrol, Δ)=1749 AFU/min. FIG. 1 d shows effects ofresveratrol on nicotinamide inhibition of SIRT1. Kinetic constants areshown relative to those of the control (no nicotinamide, no resveratrol)and represent the mean of two determinations. Error bars are standarderrors of the mean. The variable substrate in each experiment (N=NAD⁺,P=p53 acetylated peptide), the presence/absence of nicotinamide (±) andthe resveratrol concentration (μM) are indicated beneath each pair ofK_(m)−V_(max) bars.

FIG. 2 a through 2 d show the effects of polyphenols on Sir2 and S.cerevisiae lifespan. FIG. 2 a shows the initial deacetylation rate ofrecombinant GST-Sir2 as a function of resveratrol concentration. Rateswere determined at the indicated resveratrol concentrations, either with100 μM ‘Fluor de Lys’ acetylated lysine substrate (FdL) plus 3 mM NAD⁺(Δ) or with 200 μM p53-382 acetylated peptide substrate plus 200 μM NAD⁺(▪). FIG. 2 b shows lifespan analyses determined by micro-manipulatingindividual yeast cells as described Sinclair, D. A. and Guarente (Cell91, 1033-42 (1997)) in complete 2% glucose medium with 10 μM of eachcompound, unless otherwise stated. Average lifespan was determined forwild type untreated (□), quercetin (◯) and piceatannol (●). FIG. 2 cshows the average lifespan for wild type untreated (□), fisetin (◯),butein (

), or resveratrol (Δ). FIG. 2 d shows average lifespan for wild typeuntreated (□), and growth with resveratrol at 10 μM (Δ), 100 μM (●), or500 μM (◯).

FIGS. 3 a through 3 f show resveratrol extending lifespan by mimickingCR and suppressing rDNA recombination. Yeast lifespans were determinedas in FIG. 2. FIG. 3 a shows average lifespan for wild type (wt)untreated (□), wild type+resveratrol (wt+R; ●) andglucose-restricted+resveratrol (CR+R; ◯). FIG. 3 b shows averagelifespans for wild type (□), sir2(Δ) sir2+resveratrol (sir2+R; ▴), pnc1(◯), and pnc1+resveratrol (pnc1+R; ●). FIG. 3 c shows resveratrolsuppressing the frequency of ribosomal DNA recombination in the presenceand absence of nicotinamide (NAM). Frequencies were determined by lossof the ADE2 marker gene from the rDNA locus (RDN1). FIG. 3 d shows thatresveratrol does not suppress rDNA recombination in a sir2 strain. FIG.3 e show that resveratrol and other sirtuin activators do notsignificantly increase rDNA silencing compared to a 2×SIR2 strain.Pre-treated cells (RDN1::URA3) were harvested and spotted as 10-foldserial dilutions on either SC or SC with 5-fluororotic acid (5-FOA). Inthis assay, increased rDNA silencing results in increased survival on5-FOA medium. FIG. 3 f show quantitation of the effect of resveratrol onrDNA silencing by counting numbers of surviving cells on FOA/totalplated.

FIGS. 4 a through 4 e show resveratrol and other polyphenols stimulatingSIRT1 activity in human cells. FIG. 4 a shows a method for assayingintracellular deacetylase activity with a fluorogenic, cell-permeablesubstrate, FdL (‘Fluor de Lys’, BIOMOL). FdL (200 μM) is added to growthmedia and cells are incubated for 1-3 hours to allow FdL to enter thecells and the lysine-deacetylated product (deAc-FdL) to accumulateintracellularly. Cells are lysed with detergent in the presence of 1 μMTSA and 1 mM nicotinamide. Addition of the non-cell-permeable Developer(BIOMOL) releases a fluorophor, specifically from deAc-FdL. FIG. 4 bshows SIRT1 activating polyphenols stimulating TSA-insensitive, FdLdeacetylation by HeLa S3 cells. Cells were grown adherently in DMEM/10%FCS and treated for 1 hour with 200 μM FdL, 1 μM TSA and either vehicle(0.5% final DMSO, Control) or 500 μM of the indicated compound.Intracellular accumulation of deAc-FdL was then determined as describedbriefly in FIG. 4 a. The intracellular deAc-FdL level for each compound(mean of six replicates) are plotted against the ratios to the controlrate obtained in the in vitro SIRT1 polyphenol screen (see Table 1,Supplementary Tables 1 and 3). FIG. 4 c shows U2OS osteosarcoma cellsgrown to ≧90% confluence in DMEM/10% FCS exposed to 0 or 10 grays ofgamma irradiation (IR). Whole cell lysates were prepared 4 hourspost-irradiation and were probed by Western blotting with indicatedantibodies. FIG. 4 d shows U2OS cells cultured as above and pre-treatedwith the indicated amounts of resveratrol or a 0.5% DMSO blank for 4hours after which cells were exposed to 0 or 50 J/cm² of UV radiation.Lysates were prepared and analyzed by Western blot as in FIG. 4 c. FIG.4 e shows human embryonic kidney cells (HEK 293) expressing wild typeSIRT1 or dominant negative SIRT1-H363Y (SIRT1-HY) protein cultured asdescribed above, pre-treated with the indicated amounts of resveratrolor a 0.5% DMSO blank for 4 hours, and exposed to 50 J/cm² of UVradiation as above. Lysates were prepared and analyzed as above.

FIG. 5 shows the deacetylation site preferences of recombinant SIRT1.Initial rates of deacetylation were determined for a series offluorogenic acetylated peptide substrates based on short stretches ofhuman histone H3, H4 and p53 sequence. Substrates examined include:H3-4-9 with the sequence K(Ac)QTARK(Ac) (SEQ ID NO:1); H3-9-14 with thesequence K(Ac)STGGK(Ac) (SEQ ID NO:2); H3-9-14/pS with the sequenceK(Ac)—S(PO3)-TGGK(Ac) (SEQ ID NO:3); H3-14-18 with the sequenceK(Ac)APRK(Ac) (SEQ ID NO:4); H4-1-5 with the sequence SGRGK(Ac)(SEQ IDNO:5); H4-12-16(Fluor de Lys-H4-AcK16) with the sequence KGGAK(Ac) (SEQID NO:6); H4-12-16/diAc with the sequence K(Ac)GGAK(Ac)(SEQ ID NO:7);p53-320 (Fluor de Lys-SIRT2) with the sequence QPKK(Ac)(SEQ ID NO:8);p53-373 with the sequence K(Ac)SKK(Ac)(SEQ ID NO:9); p53-382(Fluor deLys-SIRT1 with the sequence RHKK(Ac) (SEQ ID NO:10); p53-382/di-Ac(Fluor de Lys-HDAC8) with the sequence RHK(Ac)K(Ac)(SEQ ID NO:11); andε-acetyl lysine (Fluor de Lys, Fdl) wit the sequence K(Ac). Allsubstrate were obtained from BIOMOL, Plymouth Meeting, Pa.). Recombinanthuman SIRT1 (1 μg, BIOMOL), was incubated for 10 minutes at 37° C. with25 μM of the indicated fluorogenic acetylated peptide substrate and 500μM NAD⁺. Reactions were stopped by the addition of 1 mM nicotinamide andthe deacetylation-dependent fluorescent signal was determined.

FIG. 6 a through 6 c show intracellular deacetylation activity measuredwith a cell-permeable, fluorogenic HDAC and sirtuin substrate. HeLa S3cells were grown to confluence in DMEM/10% FCS and then incubated withfresh medium containing 200 μM FdL for the indicated times at 37° C.Intracellular and medium levels of deacetylated substrate (deAc-FdL)were determined according to the manufacturer's instructions (HDAC assaykit, BIOMOL). All data points represent the mean of two determinations.FIG. 6 a shows the concentration ratio of intracellular ([deAc-FdL]_(i))to medium ([deAc-FdL]_(o)) concentrations in the presence (Δ) or absence(▪) of 1 μM trichostatin A (TSA). FIG. 6 b shows total accumulation ofdeacetylated substrate (deAc-FdL) in the presence (Δ) or absence (▪) of1 μM TSA. FIG. 6 c shows intracellular accumulation of deacetylatedsubstrate (deAc-FdL) in the presence (Δ) or absence (▪) of 1 μM TSA.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. “Activating a sirtuin protein”refers to the action of producing an activated sirtuin protein, i.e., asirtuin protein that is capable of performing at least one of itsbiological activities to at least some extent, e.g., with an increase ofactivity of at least about 10%, 50%, 2 fold or more. Biologicalactivities of sirtuin proteins include deacetylation, e.g., of histonesand p53; extending lifespan; increasing genomic stability; silencingtranscription; and controlling the segregation of oxidized proteinsbetween mother and daughter cells.

An “activating compound” refers to a compound that activates a sirtuinprotein. Activating compounds may have a formula selected from the groupof formulas 1-25 and 30.

A “form that is naturally occurring” when referring to a compound meansa compound that is in a form, e.g., a composition, in which it can befound naturally. For example, since resveratrol can be found in redwine, it is present in red wine in a form that is naturally occurring. Acompound is not in a form that is naturally occurring if, e.g., thecompound has been purified and separated from at least some of the othermolecules that are found with the compound in nature.

“Inhibiting a sirtuin protein” refers to the action of reducing at leastone of the biological activities of a sirtuin protein to at least someextent, e.g., at least about 10%, 50%, 2 fold or more.

An “inhibitory compound” or “inhibiting compound” refers to a compoundthat inhibits a sirtuin protein. Inhibitory compounds may have a formulaselected from the group of formulas 26-29 and 31.

A “naturally occurring compound” refers to a compound that can be foundin nature, i.e., a compound that has not been designed by man. Anaturally occurring compound may have been made by man or by nature.

“Replicative lifespan” which is used interchangeably herein with“lifespan” of a cell refers to the number of daughter cells produced byan individual “mother cell.” “Chronological aging,” on the other hand,refers to the length of time a population of non-dividing cells remainsviable when deprived of nutrients. “Increasing the lifespan of a cell”or “extending the lifespan of a cell,” as applied to cells or organisms,refers to increasing the number of daughter cells produced by one cell;increasing the ability of cells or organisms to cope with stresses andcombat damage, e.g., to DNA, proteins; and/or increasing the ability ofcells or organisms to survive and exist in a living state for longerunder a particular condition, e.g., stress. Lifespan can be increased byat least about 20%, 30%, 40%, 50%, 60% or between 20% and 70%, 30% and60%, 40% and 60% or more using methods described herein.

“Sirtuin deacetylase protein family members;” “Sir2 family members;”“Sir2 protein family members;” or “sirtuin proteins” includes yeastSir2, Sir-2.1, and human SIRT1 and SIRT2 proteins. Other family membersinclude the four additional yeast Sir2-like genes termed “HST genes”(homologues of Sir two) HST1, HST2, HST3 and HST4, and the five otherhuman homologues hSIRT3, hSIRT4, hSIRT5, hSIRT6 and hSIRT7 (Brachmann etal. (1995) Genes Dev. 9:2888 and Frye et al. (1999) BBRC 260:273).Preferred sirtuins are those that share more similarities with SIRT1,i.e., hSIRT1, and/or Sir2 than with SIRT2, such as those members havingat least part of the N-terminal sequence present in SIRT1 and absent inSIRT2 such as SIRT3 has.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “including”is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

The term “cis” is art-recognized and refers to the arrangement of twoatoms or groups around a double bond such that the atoms or groups areon the same side of the double bond. Cis configurations are oftenlabeled as (Z) configurations.

The term “trans” is art-recognized and refers to the arrangement of twoatoms or groups around a double bond such that the atoms or groups areon the opposite sides of a double bond. Trans configurations are oftenlabeled as (E) configurations.

The term “covalent bond” is art-recognized and refers to a bond betweentwo atoms where electrons are attracted electrostatically to both nucleiof the two atoms, and the net effect of increased electron densitybetween the nuclei counterbalances the internuclear repulsion. The termcovalent bond includes coordinate bonds when the bond is with a metalion.

The term “therapeutic agent” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. Examples of therapeutic agents, also referred to as “drugs”,are described in well-known literature references such as the MerckIndex, the Physicians Desk Reference, and The Pharmacological Basis ofTherapeutics, and they include, without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of a disease or illness;substances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance. The term thusmeans any substance intended for use in the diagnosis, cure, mitigation,treatment or prevention of disease or in the enhancement of desirablephysical or mental development and/or conditions in an animal or human.The phrase “therapeutically-effective amount” means that amount of sucha substance that produces some desired local or systemic effect at areasonable benefit/risk ratio applicable to any treatment. Thetherapeutically effective amount of such substance will vary dependingupon the subject and disease condition being treated, the weight and ageof the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. For example, certain compositions describedherein may be administered in a sufficient amount to produce a at areasonable benefit/risk ratio applicable to such treatment.

The term “synthetic” is art-recognized and refers to production by invitro chemical or enzymatic synthesis.

The term “meso compound” is art-recognized and refers to a chemicalcompound which has at least two chiral centers but is achiral due to aplane or point of symmetry.

The term “chiral” is art-recognized and refers to molecules that havethe property of non-superimposability of the mirror image partner, whilethe term “achiral” refers to molecules that are superimposable on theirmirror image partner. A “prochiral molecule” is a molecule that has thepotential to be converted to a chiral molecule in a particular process.

The term “stereoisomers” is art-recognized and refers to compounds thathave identical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space. In particular,“enantiomers” refer to two stereoisomers of a compound that arenon-superimposable mirror images of one another. “Diastereomers”, on theother hand, refers to stereoisomers with two or more centers ofdissymmetry and whose molecules are not mirror images of one another.

Furthermore, a “stereoselective process” is one that produces aparticular stereoisomer of a reaction product in preference to otherpossible stereoisomers of that product. An “enantioselective process” isone that favors production of one of the two possible enantiomers of areaction product.

The term “regioisomers” is art-recognized and refers to compounds thathave the same molecular formula but differ in the connectivity of theatoms. Accordingly, a “regioselective process” is one that favors theproduction of a particular regioisomer over others, e.g., the reactionproduces a statistically significant increase in the yield of a certainregioisomer.

The term “epimers” is art-recognized and refers to molecules withidentical chemical constitution and containing more than onestereocenter, but which differ in configuration at only one of thesestereocenters.

The term “ED₅₀” is art-recognized. In certain embodiments, ED₅₀ meansthe dose of a drug that produces 50% of its maximum response or effect,or alternatively, the dose that produces a pre-determined response in50% of test subjects or preparations. The term “LD₅₀” is art-recognized.In certain embodiments, LD₅₀ means the dose of a drug that is lethal in50% of test subjects. The term “therapeutic index” is an art-recognizedterm that refers to the therapeutic index of a drug, defined asLD₅₀/ED₅₀.

The term “structure-activity relationship” or “(SAR)” is art-recognizedand refers to the way in which altering the molecular structure of adrug or other compound alters its biological activity, e.g., itsinteraction with a receptor, enzyme, nucleic acid or other target andthe like.

The term “aliphatic” is art-recognized and refers to a linear, branched,cyclic alkane, alkene, or alkyne. In certain embodiments, aliphaticgroups in the present compounds are linear or branched and have from 1to about 20 carbon atoms.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure. The term “alkyl” is also defined to include halosubstitutedalkyls.

Moreover, the term “alkyl” (or “lower alkyl”) includes “substitutedalkyls”, which refers to alkyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents may include, for example, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain may themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CN and thelike. Exemplary substituted alkyls are described below. Cycloalkyls maybe further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CN, and the like.

The term “aralkyl” is art-recognized and refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to aboutten carbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The term “heteroatom” is art-recognized and refers to an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring may be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to 3- to about 10-membered ring structures, alternatively 3- toabout 7-membered rings, whose ring structures include one to fourheteroatoms. Heterocycles may also be polycycles. Heterocyclyl groupsinclude, for example, thiophene, thianthrene, furan, pyran,isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole, imidazole,pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The terms “polycyclyl” or “polycyclic group” are art-recognized andrefer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “carbocycle” is art-recognized and refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl”means —OH; and the term “sulfonyl” is art-recognized and refers to —SO₂⁻. “Halide” designates the corresponding anion of the halogens, and“pseudohalide” has the definition set forth on page 560 of “AdvancedInorganic Chemistry” by Cotton and Wilkinson.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50, R51 and R52 each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R61, or R50 and R51, taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R61 represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In certain embodiments, only oneof R50 or R51 may be a carbonyl, e.g., R50, R51 and the nitrogentogether do not form an imide. In other embodiments, R50 and R51 (andoptionally R52) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R61. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R50 and R51 is an alkylgroup.

The term “acylamino” is art-recognized and refers to a moiety that maybe represented by the general formula:

wherein R50 is as defined above, and R54 represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R61, where m and R61 are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of amidesmay not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as maybe represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and R55 andR56 represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R61 or apharmaceutically acceptable salt, R56 represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R61, where m and R61 are defined above. WhereX50 is an oxygen and R55 or R56 is not hydrogen, the formula representsan “ester”. Where X50 is an oxygen, and R55 is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR55 is a hydrogen, the formula represents a “carboxylic acid”. Where X50is an oxygen, and R56 is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X50 is asulfur and R55 or R56 is not hydrogen, the formula represents a“thiolester.” Where X50 is a sulfur and R55 is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X50 is a sulfur and R56 ishydrogen, the formula represents a “thiolformate.” On the other hand,where X50 is a bond, and R55 is not hydrogen, the above formularepresents a “ketone” group. Where X50 is a bond, and R55 is hydrogen,the above formula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R61, where m and R61 are described above.

The term “sulfonate” is art recognized and refers to a moiety that maybe represented by the general formula:

in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art recognized and includes a moiety that may berepresented by the general formula:

in which R57 is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that maybe represented by the general formula:

in which R50 and R56 are as defined above.

The term “sulfamoyl” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R50 and R51 are as defined above.

The term “sulfonyl” is art-recognized and refers to a moiety that may berepresented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term “sulfoxido” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R58 is defined above.

The term “phosphoryl” is art-recognized and may in general berepresented by the formula:

wherein Q50 represents S or O, and R59 represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl may be represented by thegeneral formulas:

wherein Q50 and R59, each independently, are defined above, and Q51represents O, S or N. When Q50 is S, the phosphoryl moiety is a“phosphorothioate”.

The term “phosphoramidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above.

The term “phosphonamidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above, and R60 represents alower alkyl or an aryl.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g. alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

The term “selenoalkyl” is art-recognized and refers to an alkyl grouphaving a substituted seleno group attached thereto. Exemplary“selenoethers” which may be substituted on the alkyl are selected fromone of —Se-alkyl, —Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R61, m andR61 being defined above.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

Certain compounds contained in compositions described herein may existin particular geometric or stereoisomeric forms. In addition, compoundsmay also be optically active. Contemplated herein are all suchcompounds, including cis- and trans-isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof,and other mixtures thereof. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are encompassed herein.

If, for instance, a particular enantiomer of a compound is desired, itmay be prepared by asymmetric synthesis, or by derivation with a chiralauxiliary, where the resulting diastereomeric mixture is separated andthe auxiliary group cleaved to provide the pure desired enantiomers.Alternatively, where the molecule contains a basic functional group,such as amino, or an acidic functional group, such as carboxyl,diastereomeric salts are formed with an appropriate optically-activeacid or base, followed by resolution of the diastereomers thus formed byfractional crystallization or chromatographic means well known in theart, and subsequent recovery of the pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Heteroatomssuch as nitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. Compounds are not intended to be limited inany manner by the permissible substituents of organic compounds.

The chemical elements are identified in accordance with the PeriodicTable of the Elements, CAS version, Handbook of Chemistry and Physics,67th Ed., 1986-87, inside cover. Also, the term “hydrocarbon” iscontemplated to include all permissible compounds having at least onehydrogen and one carbon atom. In a broad aspect, the permissiblehydrocarbons include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic organic compoundsthat may be substituted or unsubstituted.

The term “protecting group” is art-recognized and refers to temporarysubstituents that protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed by Greene and Wuts inProtective Groups in Organic Synthesis (2^(nd) ed., Wiley: N.Y., 1991).

The term “hydroxyl-protecting group” is art-recognized and refers tothose groups intended to protect a hydrozyl group against undesirablereactions during synthetic procedures and includes, for example, benzylor other suitable esters or ethers groups known in the art.

The term “carboxyl-protecting group” is art-recognized and refers tothose groups intended to protect a carboxylic acid group, such as theC-terminus of an amino acid or peptide or an acidic or hydroxyl azepinering substituent, against undesirable reactions during syntheticprocedures and includes. Examples for protecting groups for carboxylgroups involve, for example, benzyl ester, cyclohexyl ester,4-nitrobenzyl ester, t-butyl ester, 4-pyridylmethyl ester, and the like.

The term “amino-blocking group” is art-recognized and refers to a groupwhich will prevent an amino group from participating in a reactioncarried out on some other functional group, but which can be removedfrom the amine when desired. Such groups are discussed by in Ch. 7 ofGreene and Wuts, cited above, and by Barton, Protective Groups inOrganic Chemistry ch. 2 (McOmie, ed., Plenum Press, New York, 1973).Examples of suitable groups include acyl protecting groups such as, toillustrate, formyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl,methoxysuccinyl, benzyl and substituted benzyl such as3,4-dimethoxybenzyl, o-nitrobenzyl, and triphenylmethyl; those of theformula —COOR where R includes such groups as methyl, ethyl, propyl,isopropyl, 2,2,2-trichloroethyl, 1-methyl-1-phenylethyl, isobutyl,t-butyl, t-amyl, vinyl, allyl, phenyl, benzyl, p-nitrobenzyl,o-nitrobenzyl, and 2,4-dichlorobenzyl; acyl groups and substituted acylsuch as formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl,trifluoroacetyl, benzoyl, and p-methoxybenzoyl; and other groups such asmethanesulfonyl, p-toluenesulfonyl, p-bromobenzenesulfonyl,p-nitrophenylethyl, and p-toluenesulfonyl-aminocarbonyl. Preferredamino-blocking groups are benzyl (—CH₂C₆H₅), acyl [C(O)R1] or SiR1₃where R1 is C₁-C₄ alkyl, halomethyl, or 2-halo-substituted-(C₂-C₄alkoxy), aromatic urethane protecting groups as, for example,carbonylbenzyloxy (Cbz); and aliphatic urethane protecting groups suchas t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (FMOC).

The definition of each expression, e.g. lower alkyl, m, n, p and thelike, when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The term “electron-withdrawing group” is art-recognized, and refers tothe tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (σ)constant. This well known constant is described in many references, forinstance, March, Advanced Organic Chemistry 251-59 (McGraw Hill BookCompany: New York, 1977). The Hammett constant values are generallynegative for electron donating groups (σ(P)=−0.66 for NH₂) and positivefor electron withdrawing groups (σ(P)=0.78 for a nitro group), σ(P)indicating para substitution. Exemplary electron-withdrawing groupsinclude nitro, acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride,and the like. Exemplary electron-donating groups include amino, methoxy,and the like.

The term “small molecule” is art-recognized and refers to a compositionwhich has a molecular weight of less than about 2000 amu, or less thanabout 1000 amu, and even less than about 500 amu. Small molecules maybe, for example, nucleic acids, peptides, polypeptides, peptide nucleicacids, peptidomimetics, carbohydrates, lipids or other organic (carboncontaining) or inorganic molecules. Many pharmaceutical companies haveextensive libraries of chemical and/or biological mixtures, oftenfungal, bacterial, or algal extracts, which can be screened with any ofthe assays described herein. The term “small organic molecule” refers toa small molecule that is often identified as being an organic ormedicinal compound, and does not include molecules that are exclusivelynucleic acids, peptides or polypeptides.

The term “modulation” is art-recognized and refers to up regulation(i.e., activation or stimulation), down regulation (i.e., inhibition orsuppression) of a response, or the two in combination or apart.

The term “treating” is art-recognized and refers to curing as well asameliorating at least one symptom of any condition or disease.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration of a drug to a host. If it is administeredprior to clinical manifestation of the unwanted condition (e.g., diseaseor other unwanted state of the host animal) then the treatment isprophylactic, i.e., it protects the host against developing the unwantedcondition, whereas if administered after manifestation of the unwantedcondition, the treatment is therapeutic (i.e., it is intended todiminish, ameliorate or maintain the existing unwanted condition or sideeffects therefrom).

A “patient,” “subject” or “host” to be treated by the subject method maymean either a human or non-human animal.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, bovines, porcines, canines, felines, and rodents(e.g., mice and rats).

The term “bioavailable” when referring to a compound is art-recognizedand refers to a form of a compound that allows for it, or a portion ofthe amount of compound administered, to be absorbed by, incorporated to,or otherwise physiologically available to a subject or patient to whomit is administered.

The term “pharmaceutically-acceptable salts” is art-recognized andrefers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds, including, for example, those contained incompositions described herein.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof from one organ, or portion ofthe body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The terms “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” areart-recognized and refer to the administration of a subject composition,therapeutic or other material other than directly into the centralnervous system, such that it enters the patient's system and, thus, issubject to metabolism and other like processes.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articulare, subcapsular, subarachnoid, intraspinal, andintrastemal injection and infusion.

Exemplary Methods and Compositions

Provided herein are methods for activating a sirtuin deacetylase proteinfamily member (referred to as “sirtuin protein”). The methods maycomprise contacting the sirtuin deacetylase protein family member with acompound, such as a polyphenol, e.g. a plant polyphenol, and referred toherein as “activation compound” or “activating compound.” Exemplarysirtuin deacetylase proteins include the yeast silent informationregulator 2 (Sir2) and human SIRT1. Other family members includeproteins having a significant amino acid sequence homology andbiological activity, e.g., the ability to deacetylate target proteins,such as histones and p53, to those of Sir2 and SIRT1.

Exemplary activating compounds are those selected from the groupconsisting of flavones, stilbenes, flavanones, isoflavanones, catechins,chalcones, tannins and anthocyanidins. Exemplary stilbenes includehydroxystilbenes, such as trihydroxystilbenes, e.g.,3,5,4′-trihydroxystilbene (“resveratrol”). Resveratrol is also known as3,4′5-stilbenetriol. Tetrahydroxystilbenes, e.g., piceatannol, are alsoencompassed. Hydroxychalones including trihydroxychalones, such asisoliquiritigenin, and tetrahydroxychalones, such as butein, can also beused. Hydroxyflavones including tetrahydroxyflavones, such as fisetin,and pentahydroxyflavones, such as quercetin, can also be used.

In one embodiment, methods for activating a sirtuin protein comprise anactivating compound that is a stilbene or chalcone compound of formula1:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ represent H, alkyl,aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, orcarboxyl;

R represents H, alkyl, or aryl;

M represents O, NR, or S;

A-B represents a bivalent alkyl, alkenyl, alkynyl, amido, sulfonamido,diazo, ether, alkylamino, alkylsulfide, or hydrazine group; and

n is 0 or 1.

In a further embodiment, the methods comprise a compound of formula 1and the attendant definitions, wherein n is 0. In a further embodiment,the methods comprise a compound of formula 1 and the attendantdefinitions, wherein n is 1. In a further embodiment, the methodscomprise a compound of formula 1 and the attendant definitions, whereinA-B is ethenyl. In a further embodiment, the methods comprise a compoundof formula 1 and the attendant definitions, wherein A-B is—CH₂CH(Me)CH(Me)CH₂—. In a further embodiment, the methods comprise acompound of formula 1 and the attendant definitions, wherein M is O. Ina further embodiment, the methods comprises a compound of formula 1 andthe attendant definitions, wherein R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃,R′₄, and R′₅ are H. In a further embodiment, the method comprise acompound of formula 1 and the attendant definitions, wherein R₂, R₄, andR′₃ are OH. In a further embodiment, the methods comprise a compound offormula 1 and the attendant definitions, wherein R₂, R₄, R′₂ and R′₃ areOH. In a further embodiment, the methods comprise a compound of formula1 and the attendant definitions, wherein R₃, R₅, R′₂ and R′₃ are OH. Ina further embodiment, the methods comprise a compound of formula 1 andthe attendant definitions, wherein R₁, R₃, R₅, R′₂ and R′₃ are OH. In afurther embodiment, the methods comprise a compound of formula 1 and theattendant definitions, wherein R₂ and R′₂ are OH; R₄ is O-β-D-glucoside;and R′₃ is OCH₃. In a further embodiment, the methods comprise acompound of formula 1 and the attendant definitions, wherein R₂ is OH;R₄ is O-β-D-glucoside; and R′₃ is OCH₃.

In a further embodiment, the methods comprise a compound of formula 1and the attendant definitions, wherein n is 0; A-B is ethenyl; and R₁,R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ are H (trans stilbene). In afurther embodiment, the methods comprise a compound of formula 1 and theattendant definitions, wherein n is 1; A-B is ethenyl; M is O; and R₁,R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ are H (chalcone). In afurther embodiment, the methods comprise a compound of formula 1 and theattendant definitions, wherein n is 0; A-B is ethenyl; R₂, R₄, and R′₃are OH; and R₁, R₃, R₅, R′₁, R′₂, R′₄, and R′₅ are H (resveratrol). In afurther embodiment, the methods comprise a compound of formula 1 and theattendant definitions, wherein n is 0; A-B is ethenyl; R₂, R₄, R′₂ andR′₃ are OH; and R₁, R₃, R₅, R′₁, R′₄ and R′₅ are H (piceatannol). In afurther embodiment, the methods comprise a compound of formula 1 and theattendant definitions, wherein n is 1; A-B is ethenyl; M is O; R₃, R₅,R′₂ and R′₃ are OH; and R₁, R₂, R₄, R′₁, R′₄, and R′₅ are H (butein). Ina further embodiment, the methods comprise a compound of formula 1 andthe attendant definitions, wherein n is 1; A-B is ethenyl; M is O; R₁,R₃, R₅, R′₂ and R′₃ are OH; and R₂, R₄, R′₁, R′₄, and R′₅ are H(3,4,2′,4′,6′-pentahydroxychalcone). In a further embodiment, themethods comprise a compound of formula 1 and the attendant definitions,wherein n is 0; A-B is ethenyl; R₂ and R′₂ are OH, R₄ isO-β-D-glucoside, R′₃ is OCH₃; and R₁, R₃, R₅, R′₁, R′₄, and R′₅ are H(rhapontin). In a further embodiment, the methods comprise a compound offormula 1 and the attendant definitions, wherein n is 0; A-B is ethenyl;R₂ is OH, R₄ is O-β-D-glucoside, R′₃ is OCH₃; and R₁, R₃, R₅, R′₁, R′₂,R′₄, and R′₅ are H (deoxyrhapontin). In a further embodiment, themethods comprise a compound of formula 1 and the attendant definitions,wherein n is 0; A-B is —CH₂CH(Me)CH(Me)CH₂—; R₂, R₃, R′₂, and R′₃ areOH; and R₁, R₄, R₅, R′₁, R′₄, and R′₅ are H (NDGA).

In another embodiment, methods for activating a sirtuin protein comprisean activating compound that is a flavanone compound of formula 2:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, R′₅, and R″ represent H, alkyl,aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, orcarboxyl;

R represents H, alkyl, or aryl;

M represents H₂, O, NR, or S;

Z represents CR, O, NR, or S; and

X represents CR or N; and

Y represents CR or N.

In a further embodiment, the methods comprise a compound of formula 2and the attendant definitions, wherein X and Y are both CH. In a furtherembodiment, the methods comprise a compound of formula 2 and theattendant definitions, wherein M is O. In a further embodiment, themethods comprise a compound of formula 2 and the attendant definitions,wherein. M is H₂. In a further embodiment, the methods comprise acompound of formula 2 and the attendant definitions, wherein Z is O. Ina further embodiment, the methods comprise a compound of formula 2 andthe attendant definitions, wherein R″ is H. In a further embodiment, themethods comprise a compound of formula 2 and the attendant definitions,wherein R″ is OH. In a further embodiment, the methods comprise acompound of formula 2 and the attendant definitions, wherein R″ is anester. In a further embodiment, the methods comprise a compound offormula 2 and the attendant definitions, wherein R₁ is

In a further embodiment, the methods comprise a compound of formula 2and the attendant definitions, wherein R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃,R′₄, R′₅ and R″ are H. In a further embodiment, the methods comprise acompound of formula 2 and the attendant definitions, wherein R₂, R₄, andR′₃ are OH. In a further embodiment, the methods comprise a compound offormula 2 and the attendant definitions, wherein R₄, R′₂, R′₃, and R″are OH. In a further embodiment, the methods comprise a compound offormula 2 and the attendant definitions, wherein R₂, R₄, R′₂, R′₃, andR″ are OH. In a further embodiment, the methods comprise a compound offormula 2 and the attendant definitions, wherein R₂, R₄, R′₂, R′₃, R′₄,and R″ are OH.

In a further embodiment, the methods comprise a compound of formula 2and the attendant definitions, wherein X and Y are CH; M is O; Z and O;R″ is H; and R₁, R₂, R₃, R₄, R′₁, R′₂, R′₃, R′₄, R′₅ and R″ are H(flavanone). In a further embodiment, the methods comprise a compound offormula 2 and the attendant definitions, wherein X and Y are CH; M is O;Z and O; R″ is H; R₂, R₄, and R′₃ are OH; and R₁, R₃, R′₁, R′₂, R′₄, andR′₅ are H (naringenin). In a further embodiment, the methods comprise acompound of formula 2 and the attendant definitions, wherein X and Y areCH; M is O; Z and O; R″ is OH; R₂, R₄, R′₂, and R′₃ are OH; and R₁, R₃,R′₁, R′₄, and R′₅ are H (3,5,7,3′,4′-pentahydroxyflavanone). In afurther embodiment, the methods comprise a compound of formula 2 and theattendant definitions, wherein X and Y are CH; M is H₂; Z and O; R″ isOH; R₂, R₄, R′₂, and R′₃, are OH; and R₁, R₃, R′₁, R′₄ and R′₅ are H(epicatechin). In a further embodiment, the methods comprise a compoundof formula 2 and the attendant definitions, wherein X and Y are CH; M isH₂; Z and O; R″ is OH; R₂, R₄, R′₂, R′₃, and R′₄ are OH; and R₁, R₃,R′₁, and R′₅ are H (gallocatechin). In a further embodiment, the methodscomprise a compound of formula 2 and the attendant definitions, whereinX and Y are CH; M is H₂; Z and O; R″ is

R₂, R₄, R′₂, R′₃, R′₄, and R″ are OH; and R₁, R₃, R′₁, and R′₅ are H(epigallocatechin gallate).

In another embodiment, methods for activating a sirtuin protein comprisean activating compound that is an iso flavanone compound of formula 3:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, R′₅, and R″₁, represent H,alkyl, aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR,N(R)₂, or carboxyl;

R represents H, alkyl, or aryl;

M represents H₂, O, NR, or S;

Z represents CR, O, NR, or S; and

X represents CR or N; and

Y represents CR or N.

In another embodiment, methods for activating a sirtuin protein comprisean activating compound that is a flavone compound of formula 4:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅, represent H, alkyl,aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, orcarboxyl;

R″ is absent or represents H, alkyl, aryl, heteroaryl, alkaryl,heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, or carboxyl;

R represents H, alkyl, or aryl;

M represents H₂, O, NR, or S;

Z represents CR, O, NR, or S; and

X represents CR or N when R″ is absent or C when R″ is present.

In a further embodiment, the methods comprise a compound of formula 4and the attendant definitions, wherein X is C. In a further embodiment,the methods comprise a compound of formula 4 and the attendantdefinitions, wherein X is CR. In a further embodiment, the methodscomprise a compound of formula 4 and the attendant definitions, whereinZ is O. In a further embodiment, the methods comprise a compound offormula 4 and the attendant definitions, wherein M is O. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R″ is H. In a further embodiment, themethods comprise a compound of formula 4 and the attendant definitions,wherein R″ is OH. In a further embodiment, the methods comprise acompound of formula 4 and the attendant definitions, wherein R₁, R₂, R₃,R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ are H. In a further embodiment, themethods comprise a compound of formula 4 and the attendant definitions,wherein R₂, R′₂, and R′₃ are OH. In a further embodiment, the methodscomprise a compound of formula 4 and the attendant definitions, whereinR₂, R₄, R′₂, R′₃, and R′₄ are OH. In a further embodiment, the methodscomprise a compound of formula 4 and the attendant definitions, whereinR₂, R₄, R′₂, and R′₃ are OH. In a further embodiment, the methodscomprise a compound of formula 4 and the attendant definitions, whereinR₃, R′₂, and R′₃ are OH. In a further embodiment, the methods comprise acompound of formula 4 and the attendant definitions, wherein R₂, R₄,R′₂, and R′₃ are OH. In a further embodiment, the methods comprise acompound of formula 4 and the attendant definitions, wherein R₂, R′₂,R′₃, and R′₄ are OH. In a further embodiment, the methods comprise acompound of formula 4 and the attendant definitions, wherein R₂, R₄, andR′₃ are OH. In a further embodiment, the methods comprise a compound offormula 4 and the attendant definitions, wherein R₂, R₃, R₄, and R′₃ areOH. In a further embodiment, the methods comprise a compound of formula4 and the attendant definitions, wherein R₂, R₄, and R′₃ are OH. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R₃, R′₁, and R′₃ are OH. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R₂ and R′₃ are OH. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R₁, R₂, R′₂, and R′₃ are OH. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R₃, R′₁, and R′₂ are OH. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R′₃ is OH. In a further embodiment, themethods comprise a compound of formula 4 and the attendant definitions,wherein R₄ and R′₃ are OH. In a further embodiment, the methods comprisea compound of formula 4 and the attendant definitions, wherein R₂ and R₄are OH. In a further embodiment, the methods comprise a compound offormula 4 and the attendant definitions, wherein R₂, R₄, R′₁, and R′₃are OH. In a further embodiment, the methods comprise a compound offormula 4 and the attendant definitions, wherein R₄ is OH. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R₂, R₄, R′₂, R′₃, and R′₄ are OH. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R₂, R′₂, R′₃, and R′₄ are OH. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein R₁, R₂, R₄, R′₂, and R′₃ are OH.

In a further embodiment, the methods comprise a compound of formula 4and the attendant definitions, wherein X is CH; R″ is absent; Z is O; Mis O; and R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ are H(flavone). In a further embodiment, the methods comprise a compound offormula 4 and the attendant definitions, wherein X is C; R″ is OH; Z isO; M is O; R₂, R′₂, and R′₃ are OH; and R₁, R₃, R₄, R′₁, R′₄, and R′₅are H (fisetin). In a further embodiment, the methods comprise acompound of formula 4 and the attendant definitions, wherein X is CH; R″is absent; Z is O; M is O; R₂, R₄, R′₂, R′₃, and R′₄ are OH; and R₁, R₃,R′₁, and R′₅ are H (5,7,3′,4′,5′-pentahydroxyflavone). In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is CH; R″ is absent; Z is O; M is O;R₂, R₄, R′₂, and R′₃ are OH; and R₁, R₃, R′₁, R′₄, and R′₅ are H(luteolin). In a further embodiment, the methods comprise a compound offormula 4 and the attendant definitions, wherein X is C, R″ is OH; Z isO; M is O; R₃, R′₂, and R′₃ are OH; and R₁, R₂, R₄, R′₁, R′₄, and R′₅are H (3,6,3′,4′-tetrahydroxyflavone). In a further embodiment, themethods comprise a compound of formula 4 and the attendant definitions,wherein X is C, R″ is OH;Zis O; M is O; R₂, R₄, R′₂, and R′₃ are OH; andR₁, R₃, R′₁, R′₄, and R′₅ are H (quercetin). In a further embodiment,the methods comprise a compound of formula 4 and the attendantdefinitions, wherein X is CH; R″ is absent; Z is O; M is O; R₂, R′₂,R′₃, and R′₄ are OH; and R₁, R₃, R₄, R′₁, and R′₅ are H. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is C; R″ is OH; Z is O; M is O; R₂, R₄,and R′₃ are OH; and R₁, R₃, R′₁, R′₂, R′₄, and R′₅ are H. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is CH; R″ is absent; Z is O; M is O;R₂, R₃, R₄, and R′₃ are OH; and R₁, R′₁, R′₂, R′₄, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is CH; R″ is absent; Z is O; M is O;R₂, R₄, and R′₃ are OH; and R₁, R₃, R′₁, R′₂, R′₄, and R′₅ are H. In aembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is C, R″ is OH; Z is O; M is O; R₃,R′₁, and R′₃ are OH; and R₁, R₂, R₄, R′₂, R′₄, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is CH; R″ is absent; Z is O; M is O; R₂and R′₃ are OH; and R₁, R₃, R₄, R′₁, R′₂, R′₄, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is C, R″ is OH; Z is O; M is O; R₁, R₂,R′₂, and R′₃ are OH; and R₁, R₂, R₄, R′₃, R′₄, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is C; R″ is OH; Z is O; M is O; R₃,R′₁, and R′₂ are OH; and R₁, R₂, R₄; R′₃, R′₄, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is CH; R″ is absent; Z is O; M is O;R′₃ is OH; and R₁, R₂, R₃, R₄, R′₁, R′₂, R′₄, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is CH; R″ is absent; Z is O; M is O; R₄and R′₃ are OH; and R₁, R₂, R₃, R′₁, R′₂, R′₄, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is CH; R″ is absent; Z is O; M is O; R₂and R₄ are OH; and R₁, R₃, R′₁, R′₂, R′₃, R′₄, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is C; R″ is OH; Z is O; M is O; R₂, R₄,R′₁, and R′₃ are OH; and R₁, R₃, R′₂, R′₄, and R′₅ are H. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is CH; R″ is absent; Z is O; M is O; R₄is OH; and R₁, R₂, R₃, R′₁, R′₂, R′₃, R′₄, and R′₅ are H. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is C; R″ is OH; Z is O; M is O; R₂, R₄,R′₂, R′₃, and R′₄ are OH; and R₁, R₃, R′₁, and R′₅ are H. In a furtherembodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is C; R″ is OH; Z is O; M is O; R₂,R′₂, R′₃, and R′₄ are OH; and R₁, R₃, R₄, R′₁, and R′₅ are H. In afurther embodiment, the methods comprise a compound of formula 4 and theattendant definitions, wherein X is C; R″ is OH; Z is O; M is O; R₁, R₂,R₄, R′₂, and R′₃ are OH; and R₃, R′₁, R′₄, and R′₅ are H.

In another embodiment, methods for activating a sirtuin protein comprisean activating compound that is an iso flavone compound of formula 5:

wherein, independently for each occurrence,

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅, represent H, alkyl,aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, orcarboxyl;

R″ is absent or represents H, alkyl, aryl, heteroaryl, alkaryl,heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, or carboxyl;

R represents H, alkyl, or aryl;

M represents H₂, O, NR, or S;

Z represents CR, O, NR, or S; and

Y represents CR or N when R″ is absent or C when R″ is present.

In a further embodiment, the methods comprise a compound of formula 5and the attendant definitions, wherein Y is CR. In a further embodiment,the methods comprise a compound of formula 5 and the attendantdefinitions, wherein Y is CH. In a further embodiment, the methodscomprise a compound of formula 5 and the attendant definitions, whereinZ is O. In a further embodiment, the methods comprise a compound offormula 5 and the attendant definitions, wherein M is O. In a furtherembodiment, the methods comprise a compound of formula 5 and theattendant definitions, wherein R₂ and R′₃ are OH. In a furtherembodiment, the methods comprise a compound of formula 5 and theattendant definitions, wherein R₂, R₄, and R′₃ are OH.

In a further embodiment, the methods comprise a compound of formula 5and the attendant definitions, wherein Y is CH; R″ is absent; Z is O; Mis O; R₂ and R′₃ are OH; and R₁, R₃, R₄, R′₁, R′₂, R′₄, and R′₅ are H.In a further embodiment, the methods comprise a compound of formula 5and the attendant definitions, wherein Y is CH; R″ is absent; Z is O; Mis O; R₂, R₄, and R′₃ are OH; and R₁, R₃, R′₁, R′₂, R′₄, and R′₅ and H.

In another embodiment, methods for activating a sirtuin protein comprisean activating compound that is an anthocyanidin compound of formula 6:

wherein, independently for each occurrence,

R₃, R₄, R₅, R₆, R₇, R₈, R′₂, R′₃, R′₄, R′₅, and R′₆ represent H, alkyl,aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, orcarboxyl;

R represents H, alkyl, or aryl; and

A⁻ represents an anion selected from the following: Cl⁻, Br⁻, or I⁻.

In a further embodiment, the methods comprise a compound of formula 6and the attendant definitions, wherein A⁻ is Cl⁻. In a furtherembodiment, the methods comprise a compound of formula 6 and theattendant definitions, wherein R₃, R₅, R₇, and R′₄ are OH. In a furtherembodiment, the methods comprise a compound of formula 6 and theattendant definitions, wherein R₃, R₅, R₇, R′₃, and R′₄ are OH. In afurther embodiment, the methods comprise a compound of formula 6 and theattendant definitions, wherein R₃, R₅, R₇, R′₃, R′₄, and R′₅ are OH.

In a further embodiment, the methods comprise a compound of formula 6and the attendant definitions, wherein A⁻ is Cl⁻; R₃, R₅, R₇, and R′₄are OH; and R₄, R₆, R₈, R′₂, R′₃, R′₅, and R′₆ are H. In a furtherembodiment, the methods comprise a compound of formula 6 and theattendant definitions, wherein A⁻is Cl⁻; R₃, R₅, R₇, R′₃, and R′₄ areOH; and R₄, R₆, R₈, R′₂, R′₅, and R′₆ are H. In a further embodiment,the methods comprise a compound of formula 6 and the attendantdefinitions, wherein A⁻ is Cl⁻; R₃, R₅, R₇, R′₃, R′₄, and R′₅ are OH;and R₄, R₆, R₈, R′₂, and R′₆ are H.

Methods for activating a sirtuin protein may also comprise a stilbene,chalcone, or flavone compound represented by formula 7:

wherein, independently for each occurrence,

M is absent or O;

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ represent H, alkyl,aryl, heteroary alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, orcarboxyl;

R_(a) represents H or the two R_(a) form a bond;

R represents H, alkyl, or aryl; and

n is 0 or 1.

In a further embodiment, the methods comprise an activating compoundrepresented by formula 7 and the attendant definitions, wherein n is 0.In a further embodiment, the methods comprise an activating compoundrepresented by formula 7 and the attendant definitions, wherein n is 1.In a further embodiment, the methods comprise an activating compoundrepresented by formula 7 and the attendant definitions, wherein M isabsent. In a further embodiment, the methods comprise an activatingcompound represented by formula 7 and the attendant definitions, whereinM is O. In a further embodiment, the methods comprise an activatingcompound represented by formula 7 and the attendant definitions, whereinR_(a) is H. In a further embodiment, the methods comprise an activatingcompound represented by formula 7 and the attendant definitions, whereinM is O and the two R_(a) form a bond.

In a further embodiment, the methods comprise an activating compoundrepresented by formula 7 and the attendant definitions, wherein R₅ is H.In a further embodiment, the methods comprise an activating compoundrepresented by formula 7 and the attendant definitions, wherein R₅ isOH. In a further embodiment, the methods comprise an activating compoundrepresented by formula 7 and the attendant definitions, wherein R₁, R₃,and R′₃ are OH. In a further embodiment, the methods comprise anactivating compound represented by formula 7 and the attendantdefinitions, wherein R₂, R₄, R′₂, and R′₃ are OH. In a furtherembodiment, the methods comprise an activating compound represented byformula 7 and the attendant definitions, wherein R₂, R′₂, and R′₃ areOH. In a further embodiment, the methods comprise an activating compoundrepresented by formula 7 and the attendant definitions, wherein R₂ andR₄ are OH.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 7 and the attendantdefinitions, wherein n is 0; M is absent; R_(a) is H; R₅ is H; R₁, R₃,and R′₃ are OH; and R₂, R₄, R′₁, R′₂, R′₄, and R′₅ are H. In a furtherembodiment, the methods comprise an activating compound represented byformula 7 and the attendant definitions, wherein n is 1; M is absent;R_(a) is H; R₅ is H; R₂, R₄, R′₂, and R′₃ are OH; and R₁, R₃, R′₁, R′₄,and R′₅ are H. In a further embodiment, the methods comprise anactivating compound represented by formula 7 and the attendantdefinitions, wherein n is 1; M is O; the two R_(a) form a bond; R₅ isOH; R₂, R′₂, and R′₃ are OH; and R₁, R₃, R₄, R′₁, R′₄, and R′₅ are H.

Other compounds for activating sirtuin deacetylase protein familymembers include compounds having a formula selected from the groupconsisting of formulas 8-25 and 30 set forth below.

R═H, alkyl, aryl, heterocyclyl, or heteroaryl

R′═H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, or carboxy

R═H, alkyl, aryl, heterocyclyl, or heteroaryl

wherein, independently for each occurrence,

R′═H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, or carboxy

R═H, alkyl, aryl, heterocyclyl, or heteroaryl

wherein, independently for each occurrence,

L represents CR₂, O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl; and

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, or carboxy.

wherein, independently for each occurrence,

L represents CR₂, O, NR, or S;

W represents CR or N;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;

Ar represents a fused aryl or heteroaryl ring; and

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, or carboxy.

wherein, independently for each occurrence,

L represents CR₂, O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl; and

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, or carboxy.

wherein, independently for each occurrence,

L represents CR₂, O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl; and

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, or carboxy.

Methods for activating a sirtuin protein may also comprise a stilbene,chalcone, or flavone compound represented by formula 30:

wherein, independently for each occurrence,

D is a phenyl or cyclohexyl group;

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ represent H, alkyl,aryl, heteroary, alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂,carboxyl, azide, ether; or any two adjacent R or R′ groups takentogether form a fused benzene or cyclohexyl group;

R represents H, alkyl, or aryl; and

A-B represents an ethylene, ethenylene, or imine group;

provided that when A-B is ethenylene and R′₃ is H: R₃ is not OH when R₁,R₂, R₄, and R₅ are H; and R₂ and R₄ are not OMe when R₁, R₃, and R₅ areH; and R₃ is not OMe when R₁, R₂, R₄, and R₅ are H.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein D is a phenyl group.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is an ethenylene or imine group.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is an ethenylene group.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein R₂ is OH.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein R₄ is OH

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein R₂ and R₄ are OH.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein D is a phenyl group; and A-B is an ethenylenegroup.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein D is a phenyl group; A-B is an ethenylene group;and R₂ and R₄ are OH.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is Cl.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is OH.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is H.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is CH₂CH₃.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is F.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is Me.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is an azide.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is SMe.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is NO₂.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is CH(CH₃)₂.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is OMe.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; R′₂ is OH; and R′₃ is OMe.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ is OH; R₄is carboxyl; and R′₃ is OH.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is carboxyl.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ and R′₄ taken together form a fused benzene ring.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; and R₄ isOH.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OCH₂OCH₃; and R′₃ is SMe.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is carboxyl.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a cyclohexyl ring; and R₂and R₄ are OH.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; and R₃ andR₄ are OMe.

In a further embodiment, the methods include contacting a cell with anactivating compound represented by formula 30 and the attendantdefinitions, wherein A-B is ethenylene; D is a phenyl ring; R₂ and R₄are OH; and R′₃ is OH.

Exemplary activating compounds are those listed in the appended Tableshaving a ratio to control rate of more than one. A preferred compound offormula 8 is Dipyridamole; a preferred compound of formula 12 isHinokitiol; a preferred compound of formula 13 is L-(+)-Ergothioneine; apreferred compound of formula 19 is Caffeic Acid Phenol Ester; apreferred compound of formula 20 is MCI-186 and a preferred compound offormula 21 is HBED (Supplementary Table 6).

Also included are pharmaceutically acceptable addition salts andcomplexes of the compounds of formulas 1-25 and 30. In cases wherein thecompounds may have one or more chiral centers, unless specified, thecompounds contemplated herein may be a single stereoisomer or racemicmixtures of stereoisomers.

In cases in which the compounds have unsaturated carbon-carbon doublebonds, both the cis (Z) and trans (E) isomers are contemplated herein.In cases wherein the compounds may exist in tautomeric forms, such asketo-enol tautomers, such as

and

each tautomeric form is contemplated as being included within themethods presented herein, whether existing in equilibrium or locked inone form by appropriate substitution with R′. The meaning of anysubstituent at any one occurrence is independent of its meaning, or anyother substituent's meaning, at any other occurrence.

Also included in the methods presented herein are prodrugs of thecompounds of formulas 1-25 and 30. Prodrugs are considered to be anycovalently bonded carriers that release the active parent drug in vivo.

Analogs and derivatives of the above-described compounds can also beused for activating a member of the sirtuin protein family. For example,derivatives or analogs may make the compounds more stable or improvetheir ability to traverse cell membranes or being phagocytosed orpinocytosed. Exemplary derivatives include glycosylated derivatives, asdescribed, e.g., in U.S. Pat. No. 6,361,815 for resveratrol. Otherderivatives of resveratrol include cis- and trans-resveratrol andconjugates thereof with a saccharide, such as to form a glucoside (see,e.g., U.S. Pat. No. 6,414,037). Glucoside polydatin, referred to aspiceid or resveratrol 3-O-beta-D-glucopyranoside, can also be used.Saccharides to which compounds may be conjugated include glucose,galactose, maltose, lactose and sucrose. Glycosylated stilbenes arefurther described in Regev-Shoshani et al. Biochemical J. (published onApr. 16, 2003 as BJ20030141). Other derivatives of compounds describedherein are esters, amides and prodrugs. Esters of resveratrol aredescribed, e.g., in U.S. Pat. No. 6,572,882. Resveratrol and derivativesthereof can be prepared as described in the art, e.g., in U.S. Pat. Nos.6,414,037; 6,361,815; 6,270,780; 6,572,882; and Brandolini et al. (2002)J. Agric. Food. Chem.50:7407. Derivatives of hydroxyflavones aredescribed, e.g., in U.S. Pat. No. 4,591,600. Resveratrol and otheractivating compounds can also be obtained commercially, e.g., fromSigma.

In certain embodiments, if an activating compound occurs naturally, itmay be at least partially isolated from its natural environment prior touse. For example, a plant polyphenol may be isolated from a plant andpartially or significantly purified prior to use in the methodsdescribed herein. An activating compound may also be preparedsynthetically, in which case it would be free of other compounds withwhich it is naturally associated. In an illustrative embodiment, anactivating composition comprises, or an activating compound isassociated with, less than about 50%, 10%, 1%, 0.1%, 10⁻²% or 10⁻³% of acompound with which it is naturally associated.

Sirtuin proteins may be activated in vitro, e.g., in a solution or in acell. In one embodiment, a sirtuin protein is contacted with anactivating compound in a solution. A sirtuin is activated by a compoundwhen at least one of its biological activities, e.g., deacetylationactivity, is higher in the presence of the compound than in its absence.Activation may be by a factor of at least about 10%, 30%, 50%, 100%(i.e., a factor of two), 3, 10, 30, or 100. The extent of activation canbe determined, e.g., by contacting the activated sirtuin with adeacetylation substrate and determining the extent of deacetylation ofthe substrate, as further described herein. The observation of a lowerlevel of acetylation of the substrate in the presence of a test sirtuinrelative to the presence of a non activated control sirtuin indicatesthat the test sirtuin is activated. The solution may be a reactionmixture. The solution may be in a dish, e.g., a multiwell dish. Sirtuinproteins may be prepared recombinantly or isolated from cells accordingto methods known in the art.

In another embodiment, a cell comprising a sirtuin deacetylase proteinis contacted with an activating compound. The cell may be a eukaryoticcell, e.g., a mammalian cell, such as a human cell, a yeast cell, anon-human primate cell, a bovine cell, an ovine cell, an equine cell, aporcine cell, a sheep cell, a bird (e.g., chicken or fowl) cell, acanine cell, a feline cell or a rodent (mouse or rat) cell. It can alsobe a non-mammalian cell, e.g., a fish cell. Yeast cells include S.cerevesiae and C. albicans. The cell may also be a prokaryotic cell,e.g., a bacterial cell. The cell may also be a single-celledmicroorganism, e.g., a protozoan. The cell may also be a metazoan cell,a plant cell or an insect cell. The application of the methods decribedherein to a large number of cell types is based at least on the highconvervation of sirtuins from humans to fungi, protozoans, metazoans andplants.

In one embodiment, the cells are in vitro. A cell may be contacted witha solution having a concentration of an activating compound of less thanabout 0.1 μM; 0.5 μM; less than about 1 μM; less than about 10 μM orless than about 100 μM. The concentration of the activating compound mayalso be in the range of about 0.1 to 1 μM, about 1 to 10 μM or about 10to 100 μM. The appropriate concentration may depend on the particularcompound and the particular cell used as well as the desired effect. Forexample, a cell may be contacted with a “sirtuin activating”concentration of an activating compound, e.g., a concentrationsufficient for activating the sirtuin by a factor of at least 10%, 30%,50%, 100%, 3, 10, 30, or 100.

In certain embodiments, a cell is contacted with an activating compoundin vivo, such as in a subject. The subject can be a human, a non-humanprimate, a bovine, an ovine, an equine, a porcine, a sheep, a canine, afeline or a rodent (mouse or rat). For example, an activating compoundmay be administered to a subject. Administration may be local, e.g.,topical, parenteral, oral, or other depending on the desired result ofthe administration (as further described herein). Administration may befollowed by measuring a factor in the subject, such as measuring theactivity of the sirtuin. In an illustrative embodiment, a cell isobtained from a subject following administration of an activatingcompound to the subject, such as by obtaining a biopsy, and the activityof the sirtuin is determined in the biopsy. The cell may be any cell ofthe subject, but in cases in which an activating compound isadministered locally, the cell is preferably a cell that is located inthe vicinity of the site of administration.

Also provided are methods for modulating the acetylation level of p53proteins. As shown herein (see, e.g., the Examples), lysine 382 of p53proteins in cells is deacetylated following incubation of cells in thepresence of low concentrations of resveratrol. Accordingly, “p53deacetylating concentrations” of compounds include, e.g., concentrationsof less than about 0.1 μM, 0.5 μM, 1 μM, 3 μM, 50 μM, 100 μM or 300 μM.It has also been shown herein that p53 proteins in cells are acetylatedin the presence of higher concentrations of resveratrol. Accordingly,“p53 acetylating concentrations” of compounds include, e.g.,concentrations of at least about 10 μM, 30 μM, 100 μM or 300 μM. Thelevel of acetylation of p53 can be determined by methods known in theart, e.g., as further described in the Examples.

Other methods contemplated are methods for protecting a cell againstapoptosis. Without wanting to be limited to a particular mechanism ofaction, but based at least in part on the fact that acetylation of p53proteins activates p53 proteins and that activated p53 proteins induceapoptosis, incubating cells comprising p53 proteins in the presence of ap53 deacetylating concentration of an activating compound prevents theinduction of apoptosis of the cells. Accordingly, a cell can beprotected from apoptosis by activating sirtuins by contacting the cellwith an amount of an activating compound sufficient or adequate forprotecting against apoptosis, e.g., less than about 0.1 μM, 0.5 μM, 1μM, 3 μM or 10 μM. An amount sufficient or adequate for protectionagainst apoptosis can also be determined experimentally, such as byincubating a cell with different amounts of an activating compound,subjecting the cell to an agent or condition that induces apoptosis, andcomparing the extent of apoptosis in the presence of differentconcentrations or the absence of an enhancing compound and determiningthe concentration that provides the desired protection. Determining thelevel of apoptosis in a population of cells can be performed accordingto methods known in the art.

Yet other methods contemplated herein are methods for inducing apoptosisin a cell. Without wanting to be limited to a particular mechanism ofaction, as shown in the Examples, at certain concentrations ofcompounds, p53 proteins are acetylated rather than deacetylated, therebyactivating the p53 proteins, and inducing apoptosis. Apoptosis inducingconcentrations of compounds may be, e.g., at least about 10 μM, 30 μM,100 μM or 300 μM.

Appropriate concentrations for modulating p53 deacetylation andapoptosis can be determined according to methods, e.g., those describedherein. Concentrations may vary slightly from one cell to another, fromone activating compound to another and whether the cell is isolated orin an organism.

Cells in which p53 acetylation and apoptosis may be modulated can be invitro, e.g., in cell culture, or in vivo, e.g., in a subject.Administration of an activating compound to a subject can be conductedas further described herein. The level of p53 acetylation and/orapoptosis in cells of the subject can be determined, e.g., by obtaininga sample of cells from the subject and conducting an in vitro analysisof the level of p53 acetylation and/or apoptosis.

Also provided herein are methods for extending the lifespan of aeukaryotic cells and/or increasing their resistance to stresscomprising, e.g., contacting the eukaryotic cell with a compound, e.g.,a polyphenol compound. Exemplary compounds include the activatingcompounds described herein, such as compounds of the stilbene, flavoneand chalcone families. Although the Examples show that quercetin andpiceatannol, which activate sirtuins, were not found to significantlyaffect the lifespan of eukaryotic cells, it is believed that this may bethe result of a lack of entry of the compounds into the cell orpotentially the existence of another pathway overriding activation ofsirtuins. Derivatives and analogs of these compounds or administrationof these compound to other cells or by other methods are expected toactivate sirtuins.

In one embodiment, methods for extending the lifespan of a eukaryoticcell and/or increasing its resistance to stress comprise contacting thecell with a stilbene, chalcone, or flavone compound represented byformula 7:

wherein, independently for each occurrence,

M is absent or O;

R₁, R₂, R₃, R₄, R₅, R′₁, R′₂, R′₃, R′₄, and R′₅ represent H, alkyl,aryl, heteroary alkaryl, heteroaralkyl, halide, NO₂, SR, OR, N(R)₂, orcarboxyl;

R_(a) represents H or the two R_(a) form a bond;

R represents H, alkyl, or aryl; and

n is 0 or 1.

In a further embodiment, the methods comprise a compound represented byformula 7 and the attendant definitions, wherein n is 0. In a furtherembodiment, the methods comprise a compound represented by formula 7 andthe attendant definitions, wherein n is 1. In a further embodiment, themethods comprise a compound represented by formula 7 and the attendantdefinitions, wherein M is absent. In a further embodiment, the methodscomprise a compound represented by formula 7 and the attendantdefinitions, wherein M is O. In a further embodiment, the methodscomprise a compound represented by formula 7 and the attendantdefinitions, wherein R_(a) is H. In a further embodiment, the methodscomprise a compound represented by formula 7 and the attendantdefinitions, wherein M is O and the two R_(a) form a bond. In a furtherembodiment, the methods comprise a compound represented by formula 7 andthe attendant definitions, wherein R₅ is H. In a further embodiment, themethods comprise a compound represented by formula 7 and the attendantdefinitions, wherein R₅ is OH. In a further embodiment, the methodscomprise a compound represented by formula 7 and the attendantdefinitions, wherein R₁, R₃, and R′₃ are OH. In a further embodiment,the methods comprise a compound represented by formula 7 and theattendant definitions, wherein R₂, R₄, R′₂, and R′₃ are OH. In a furtherembodiment, the methods comprise a compound represented by formula 7 andthe attendant definitions, wherein R₂, R′₂, and R′₃ are OH.

In a further embodiment, methods for extending the lifespan of aeukaryotic cell comprise contacting the cell with a compound representedby formula 7 and the attendant definitions, wherein n is 0; M is absent;R_(a) is H; R₅ is H; R₁, R₃, and R′₃ are OH; and R₂, R₄, R′₁, R′₂, R′₄,and R′₅ are H. In a further embodiment, the methods comprise a compoundrepresented by formula 7 and the attendant definitions, wherein n is 1;M is absent; R_(a) is H; R₅ is H; R₂, R₄, R′₂, and R′₃ are OH; and R₁,R₃, R′₁, R′₄, and R′₅ are H. In a further embodiment, the methodscomprise a compound represented by formula 7 and the attendantdefinitions, wherein n is 1; M is O; the two R_(a) form a bond; R₅ isOH; R₂, R′₂, and R′₃ are OH; and R₁, R₃, R₄, R′₁, R′₄, and R′₅ are H.

The eukaryotic cell whose lifespan may be extended can be a human, anon-human primate, a bovine, an ovine, an equine, a porcine, a sheep, acanine, a feline, a rodent (mouse or rat) or a yeast cell. A yeast cellmay be Saccharomyces cerevisiae or Candida albicans. Concentrations ofcompounds for this purpose may be about 0.1 μM, 0.3 μM, 0.5 μM, 1 μM, 3μM, 10 μM, 30 μM, 100 μM or 300 μM. Based at least on the highconservation of Sir2 proteins in various organisms, lifespan can also beprolonged in prokaryotes, protozoans, metazoans, insects and plants.

The cell may be in vitro or in vivo. In some embodiments, alife-extending compound is administered to an organism (e.g., a subject)such as to induce hormesis, i.e., an increasing resistance to mildstress that results in increasing the lifespan of the organism. In fact,it has been shown that SIR2 is essential for the increased longevityprovided by calorie restriction, a mild stress, that extends thelifespan of every organism it has been tested on (Lin et al. (2000)Science 249:2126). For example, overexpression of a Caenorhabditis.elegans SIR2 homologue, sir-2.1, increases lifespan via a forkheadtranscription factor, DAF-16, and a SIR2 gene has recently beenimplicated in lifespan regulation in Drosophila melanogaster (Rogina etal. Science (2002) 298:1745). Furthermore, the closest human Sir2homologue, SIRT1, promotes survival in human cells by down-regulatingthe activity of the tumor suppressor p53 (Tissenbaum et al. Nature 410,227-30 (2001); Rogina et al. Science, in press (2002); and Vaziri, H. etal. Cell 107, 149-59. (2001)). The role of SIR2 in stress resistance andcell longevity is further supported by the identification of PNC1 as acalorie restriction- and stress-responsive gene that increases lifespanand stress resistance of cells by depleting intracellular nicotinamide(Anderson et al. (2003) Nature 423:181 and Bitterman et al. (2002) J.Biol. Chem. 277: 45099). Accordingly, compounds may be administered to asubject for protecting the cells of the subject from stresses andthereby extending the lifespan of the cells of the subject.

Also encompassed are methods for inhibiting sirtuins; inhibitingdeacetylation of p53, e.g., for stimulating acetylation of p53;stimulating apoptosis; reducing lifespan and/or rendering cells ororganisms more sensitive to stresses. Methods may include contacting acell or a molecule, such as a sirtuin or a p53 protein, with a compoundthat inhibits sirtuins, i.e., an “inhibiting compound,” such, a compoundhaving a formula selected from the group of formulas 26-29 and 31:

wherein, independently for each occurrence,

R′ represents H, halogen, NO₂, SR, OR, NR₂, alkyl, aryl, or carboxy;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl; and

R″ represents alkyl, alkenyl, or alkynyl.

wherein, independently for each occurrence,

L represents O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;

R′ represents H, halogen, NO₂, SR, SO₃, OR, NR₂, alkyl, aryl, orcarboxy;

a represents an integer from 1 to 7 inclusively; and

b represents an integer from 1 to 4 inclusively.

wherein, independently for each occurrence,

L represents O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;

R′ represents H, halogen, NO₂, SR, SO₃, OR, NR₂, alkyl, aryl, orcarboxy;

a represents an integer from 1 to 7 inclusively; and

b represents an integer from 1 to 4 inclusively.

wherein, independently for each occurrence,

L represents O, NR, or S;

R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;

R′ represents H, halogen, NO₂, SR, SO₃, OR, NR₂, alkyl, aryl, orcarboxy;

a represents an integer from 1 to 7 inclusively; and

b represents an integer from 1 to 4 inclusively.

wherein, independently for each occurrence,

R₂, R₃, and R₄ are H, OH, or O-alkyl;

R′₃ is H or NO₂; and

A-B is an ethenylene or amido group.

In a further embodiment, the inhibiting compound is represented byformula 31 and the attendant definitions, wherein R₃ is OH, A-B isethenylene, and R′₃ is H.

In a further embodiment, the inhibiting compound is represented byformula 31 and the attendant definitions, wherein R₂ and R₄ are OH, A-Bis an amido group, and R′₃ is H.

In a further embodiment, the inhibiting compound is represented byformula 31 and the attendant definitions, wherein R₂ and R₄ are OMe, A-Bis ethenylene, and R′₃ is NO₂.

In a further embodiment, the inhibiting compound is represented byformula 31 and the attendant definitions, wherein R₃ is OMe, A-B isethenylene, and R′₃ is H.

Also included are pharmaceutically acceptable addition salts andcomplexes of the compounds of formulas 26-29 and 31. In cases whereinthe compounds may have one or more chiral centers, unless specified, thecompounds contemplated herein may be a single stereoisomer or racemicmixtures of stereoisomers.

Exemplary inhibitory compounds are those set forth in the appendedTables for which the “ratio to control rate” is lower than one.

In cases in which the compounds have unsaturated carbon-carbon doublebonds, both the cis (Z) and trans (E) isomers are contemplated herein.In cases wherein the compounds may exist in tautomeric forms, such asketo-enol tautomers, such as

and

each tautomeric form is contemplated as being included within themethods presented herein, whether existing in equilibrium or locked inone form by appropriate substitution with R′. The meaning of anysubstituent at any one occurrence is independent of its meaning, or anyother substituent's meaning, at any other occurrence.

Also included in the methods presented herein are prodrugs of thecompounds of formulas 26-29 and 31. Prodrugs are considered to be anycovalently bonded carriers that release the active parent drug in vivo.

Inhibitory compounds may be contacted with a cell, administered to asubject, or contacted with one or more molecules, such as a sirtuinprotein and a p53 protein. Doses of inhibitory compounds may be similarto those of activating compounds.

Whether in vitro or in vivo, a cell may also be contacted with more thanone compound (whether an activating compound or an inhibiting compound).A cell may be contacted with at least 2, 3, 5, or 10 differentcompounds. A cell may be contacted simultaneously or sequentially withdifferent compounds.

Also encompassed are compositions comprising one or more activating orinhibiting compounds having a formula selected from the group offormulas 1-31. Compounds may be in a pharmaceutical composition, such asa pill or other formulation for oral administration, further describedherein. Compositions may also comprise or consist of extracts of plants,red wine or other source of the compounds.

Yet other methods contemplated herein include sceening methods foridentifying compounds that modulate sirtuins. Assays may be conducted ina cell based or cell free format. For example, an assay may compriseincubating (or contacting) a sirtuin with a test compound underconditions in which a sirtuin can be activated by an agent known toactivate the sirtuin, and monitoring or determining the level ofactivation of the sirtuin in the presence of the test compound relativeto the absence of the test compound. The level of activation of asirtuin can be determined by determining its ability to deacetylate asubstrate. Exemplary substrates are acetylated peptides, e.g., those setforth in FIG. 5, which can be obtained from BIOMOL (Plymouth Meeting,Pa.). Preferred substrates include peptides of p53, such as thosecomprising an acetylated K382. A particularly preferred substrate is theFluor de Lys-SIRT1 (BIOMOL), i.e., the acetylated peptideArg-His-Lys-Lys. Other substrates are peptides from human histones H3and H4 or an acetylated amino acid (see FIG. 5). Substrates may befluorogenic. The sirtuin may be SIRT1 or Sir2 or a portion thereof. Forexample, recombinant SIRT1 can be obtained from BIOMOL. The reaction maybe conducted for about 30 minutes and stopped, e.g., with nicotinamide.The HDAC fluorescent activity assay/drug discovery kit (AK-500, BIOMOLResearch Laboratories) may be used to determine the level ofacetylation. Similar assays are described in Bitterman et al. (2002) J.Biol. Chem. 277:45099. The level of activation of the sirtuin in anassay may be compared to the level of activation of the sirtuin in thepresence of one or more (separately or simultaneously) compoundsdescribed herein, which may serve as positive or negative controls.Sirtuins for use in the assays may be full length sirtuin proteins orportions thereof. Since it has been shown herein that activatingcompounds appear to interact with the N-terminus of SIRT1, proteins foruse in the assays include N-terminal portions of sirtuins, e.g., aboutamino acids 1-176 or 1-255 of SIRT1; about amino acids 1-174 or 1-252 ofSir2.

In one embodiment, a screening assay comprises (i) contacting a sirtuinwith a test compound and an acetylated substrate under conditionsappropriate for the sirtuin to deacetylate the substrate in the absenceof the test compound; and (ii) determining the level of acetylation ofthe substrate, wherein a lower level of acetylation of the substrate inthe presence of the test compound relative to the absence of the testcompound indicates that the test compound stimulates deacetylation bythe sirtuin, whereas a higher level of acetylation of the substrate inthe presence of the test compound relative to the absence of the testcompound indicates that the test compound inhibits deacetylation by thesirtuin.

Methods for identifying compounds that modulate, e.g., stimulate orinhibit, sirtuins in vivo may comprise (i) contacting a cell with a testcompound and a substrate that is capable of entering a cell in thepresence of an inhibitor of class I and class II HDACs under conditionsappropriate for the sirtuin to deacetylate the substrate in the absenceof the test compound; and (ii) determining the level of acetylation ofthe substrate, wherein a lower level of acetylation of the substrate inthe presence of the test compound relative to the absence of the testcompound indicates that the test compound stimulates deacetylation bythe sirtuin, whereas a higher level of acetylation of the substrate inthe presence of the test compound relative to the absence of the testcompound indicates that the test compound inhibits deacetylation by thesirtuin. A preferred substrate is an acetylated peptide, which is alsoprefeably fluorogenic, as further described herein (Examples). Themethod may further comprise lysing the cells to determine the level ofacetylation of the substrate. Substrates may be added to cells at aconcentration ranging from about 1 μM to about 10 mM, preferably fromabout 10 μM to 1 mM, even more preferably from about 100 μM to 1 mM,such as about 200 μM. A preferred substrate is an acetylated lysine,e.g., ε-acetyl lysine (Fluor de Lys, FdL) or Fluor de Lys-SIRT1. Apreferred inhibitor of class I and class II HDACs is trichostatin A(TSA), which may be used at concentrations ranging from about 0.01 to100 μM, preferably from about 0.1 to 10 μM, such as 1 μM. Incubation ofcells with the test compound and the substrate may be conducted forabout 10 minutes to 5 hours, preferably for about 1-3 hours. Since TSAinhibits all class I and class II HDACs, and that certain substrates,e.g., Fluor de Lys, is a poor substrate for SIRT2 and even less asubstrate for SIRT3-7, such an assay may be used to identify modulatorsof SIRT1 in vivo. An exemplary assay is further described in theExamples and shown in FIG. 4 a.

Also provided herein are assays for identifying agents that are capableof extending or reducing the lifespan of cells and/or increasing ordecreasing their resistance to stress. A method may comprise incubatingcells with a test compound and determining the effect of the testcompound on rDNA silencing and rDNA recombination, wherein an increasein the frequency of rDNA recombination and an absence of effect on rDNAsilencing in the presence of the test compound relative to the absenceof the test compound indicates that the test compound extends lifespan.This assay is based at least on the observation that resveratrol reducedthe frequency of rDNA recombination by about 60% in a SIR2 dependentmanner, but did not increasing rDNA silencing.

Also provided herein are methods for identifying the binding site ofactivating or inhibitory compounds in sirtuin proteins. In oneembodiment, BML-232 (Table 10) is used. BML-232, has very similar SIRT1activating properties to resveratrol and contains a phenylazidefunction. Phenylazide groups may be activated by the absorption ofultraviolet light to form reactive nitrenes. When a protein-boundphenylazide is light-activated it can react to form covalent adductswith various protein functional groups in the site to which it is bound.The photo cross-linked protein may then be analyzed by proteolysis/massspectrometry to identify amino acid residues which may form part of thebinding site for the compound. This information, in combination withpublished three dimensional structural information on SIRT1 homologscould be used to aid the design of new, possibly higher affinity, SIRT1activating ligands.

Exemplary Uses

In one embodiment, cells are treated in vitro as described herein toextend their lifespan, e.g., to keep them proliferating longer and/orincreasing its resistance to stress or prevent apoptosis. That compoundsdescribed herein may increase resistance to stress is based at least onthe observation that Sir2 provides stress resistance and that PNC1modulates Sir2 activity in response to cell stress (Anderson et al.(2003) Nature 423:181). This is particularly useful for primary cellcultures (i.e., cells obtained from an organism, e.g., a human), whichare known to have only a limited lifespan in culture. Treating suchcells according to methods described herein, e.g., by contacting themwith an activating or lifespan extending compound, will result inincreasing the amount of time that the cells are kept alive in culture.Embryonic stem (ES) cells and pluripotent cells, and cellsdifferentiated therefrom, can also be treated according to the methodsdescribed herein such as to keep the cells or progeny thereof in culturefor longer periods of time. Primary cultures of cells, ES cells,pluripotent cells and progeny thereof can be used, e.g., to identifycompounds having particular biological effects on the cells or fortesting the toxicity of compounds on the cells (i.e., cytotoxicityassays). Such cells can also be used for transplantation into a subject,e.g., after ex vivo modification.

In other embodiments, cells that are intended to be preserved for longperiods of time are treated as described herein. The cells can be cellsin suspension, e.g., blood cells, serum, biological growth media, ortissues or organs. For example, blood collected from an individual foradministering to an individual can be treated as described herein, suchas to preserve the blood cells for longer periods of time, such as forforensic purposes. Other cells that one may treat for extending theirlifespan or protect against apoptosis include cells for consumption,e.g., cells from non-human mammals (such as meat), or plant cells (suchas vegetables).

Compounds may also be applied during developmental and growth phases inmammals, plants, insects or microorganisms, in order to, e.g., alter,retard or accelerate the developmental and/or growth process.

In another embodiment, cells obtained from a subject, e.g., a human orother mammal, are treated according to methods described herein and thenadministered to the same or a different subject. Accordingly, cells ortissues obtained from a donor for use as a graft can be treated asdescribed herein prior to administering to the recipient of the graft.For example, bone marrow cells can be obtained from a subject, treatedex vivo, e.g., to extend their lifespan, and then administered to arecipient. The graft can be an organ, a tissue or loose cells.

In yet other embodiments, cells are treated in vivo, e.g., to increasetheir lifespan or prevent apoptosis. For example, skin can be protectedfrom aging, e.g., developing wrinkles, by treating skin, e.g.,epithelial cells, as described herein. In an exemplary embodiment, skinis contacted with a pharmaceutical or cosmetic composition comprising acompound described herein. Exemplary skin afflictions or skin conditionsinclude disorders or diseases associated with or caused by inflammation,sun damage or natural aging. For example, the compositions find utilityin the prevention or treatment of contact dermatitis (including irritantcontact dermatitis and allergic contact dermatitis), atopic dermatitis(also known as allergic eczema), actinic keratosis, keratinizationdisorders (including eczema), epidermolysis bullosa diseases (includingpenfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas(including erythema multiforme and erythema nodosum), damage caused bythe sun or other light sources, discoid lupus erythematosus,dermatomyositis, skin cancer and the effects of natural aging. Theformulations may be administered topically, to the skin or mucosaltissue, as an ointment, lotion, cream, microemulsion, gel, solution orthe like, as described in the preceding section, within the context of adosing regimen effective to bring about the desired result. A dose ofactive agent may be in the range of about 0.005 to about 1 micromolesper kg per day, preferably about 0.05 to about 0.75 micromoles per kgper day, more typically about 0.075 to about 0.5 micromoles per kg perday. It will be recognized by those skilled in the art that the optimalquantity and spacing of individual dosages will be determined by thenature and extent of the condition being treated, the site ofadministration, and the particular individual undergoing treatment, andthat such optimums can be determined by conventional techniques. Thatis, an optimal dosing regimen for any particular patient, i.e., thenumber and frequency of doses, can be ascertained using conventionalcourse of treatment determination tests. Generally, a dosing regimenherein involves administration of the topical formulation at least oncedaily, and preferably one to four times daily, until symptoms havesubsided.

Topical formulations may also be used as chemopreventive compositions.When used in a chemopreventive method, susceptible skin is treated priorto any visible condition in a particular individual.

Compounds can also be delivered locally, e.g., to a tissue or organwithin a subject, such as by injection, e.g., to extend the lifespan ofthe cells; protect against apoptosis or induce apoptosis.

In yet another embodiment, a compound is administered to a subject, suchas to generally increase the lifespan of its cells and to protect itscells against stress and/or against apoptosis. It is believed thattreating a subject with a compound described herein is similar tosubjecting the subject to hormesis, i.e., mild stress that is beneficialto organisms and may extend their lifespan. For example, a compound canbe taken by subjects as a food or dietary supplement. In one embodiment,such a compound is a component of a multi-vitamin complex. Compounds canalso be added to existing formulations that are taken on a daily basis,e.g., statins and aspirin. Compounds may also be used as food additives.

Compounds described herein could also be taken as one component of amulti-drug complex or as a supplement in addition to a multi-drugregimen. In one embodiment, this multi-drug complex or regimen wouldinclude drugs or compounds for the treatment or prevention ofaging-related diseases, e.g., stroke, heart disease, arthritis, highblood pressure, Alzheimer's. In another embodiment, this multi-drugregimen would include chemotherapeutic drugs for the treatment ofcancer. In a specific embodiment, a polyphenol compound could be used toprotect non-cancerous cells from the effects of chemotherapy.

Compounds may be administered to subject to prevent aging andaging-related consequences or diseases, such as stroke, heart disease,arthritis, high blood pressure, and Alzheimer's disease. Compoundsdescribed herein can also be administered to subjects for treatment ofdiseases, e.g., chronic diseases, associated with cell death, such as toprotect the cells from cell death. Exemplary diseases include thoseassociated with neural cell death or muscular cell death, such asParkinson's disease, Alzheimer's disease, multiple sclerosis,amniotropic lateral sclerosis, and muscular dystrophy; AIDS; fulminanthepatitis; diseases linked to degeneration of the brain, such asCreutzfeld-Jakob disease, retinitis pigmentosa and cerebellardegeneration; myelodysplasis such as aplastic anemia; ischemic diseasessuch as myocardial infarction and stroke; hepatic diseases such asalcoholic hepatitis, hepatitis B and hepatitis C; joint-diseases such asosteoarthritis; atherosclerosis; alopecia; damage to the skin due to UVlight; lichen planus; atrophy of the skin; cataract; graft rejections;and etc.

Compounds described herein can also be administered to a subjectsuffering from an acute disease, e.g., damage to an organ or tissue,e.g., a subject suffering from stroke or myocardial infarction or asubject suffering from a spinal cord injury. Compounds can also be usedto repair an alcoholic's liver.

Compounds can also be administered to subjects who have recentlyreceived or are likely to receive a dose of radiation. In oneembodiment, the dose of radiation is received as part of a work-relatedor medical procedure, e.g., working in a nuclear power plant, flying anairplane, an X-ray, CAT scan, or the administration of a radioactive dyefor medical imaging; in such an embodiment, the compound is administeredas a prophylactic measure. In another embodiment, the radiation exposureis received unintentionally, e.g., as a result of an industrialaccident, terrorist act, or act of war involving radioactive material.In such a case, the compound is preferably administered as soon aspossible after the exposure to inhibit apoptosis and the subsequentdevelopment of acute radiation syndrome.

Based at least on the discovery that certain concentrations ofactivating compounds prevent deacetylation of p53 in cells and therebymay induce apoptosis in cells, the activating compounds can also beadministed to a subject in conditions in which apoptosis of certaincells is desired. For example, cancer may be treated or prevented.Exemplary cancers are those of the brain and kidney; hormone-dependentcancers including breast, prostate, testicular, and ovarian cancers;lymphomas, and leukemias. In cancers associated with solid tumors, aactivating compound may be administered directly into the tumor. Cancerof blood cells, e.g., leukemia can be treated by administering aactivating compound into the blood stream or into the bone marrow.Benign cell growth can also be treated, e.g., warts. Other diseases thatcan be treated include autoimmune diseases, e.g., systemic lupuserythematosus, scleroderma, and arthritis, in which autoimmune cellsshould be removed. Viral infections such as herpes, HIV, adenovirus, andHTLV-1 associated malignant and benign disorders can also be treated byadministration of compounds. Alternatively, cells can be obtained from asubject, treated ex vivo to remove certain undesirable cells, e.g.,cancer cells, and administered back to the same or a different subject.

In other embodiments, methods described herein are applied to yeastcells. Situations in which it may be desirable to extend the lifespan ofyeast cells include any process in which yeast is used, e.g., the makingof beer, yogurt, and bakery items, e.g., bread. Use of yeast having anextended lifespan can result in using less yeast or in having the yeastbe active for longer periods of time. Yeast or other mammalian cellsused for recombinantly producing proteins may also be treated asdescribed herein.

Subjects that may be treated as described herein include eukaryotes,such as mammals, e.g., humans, ovines, bovines, equines, porcines,canines, felines, non-human primate, mice, and rats. Cells that may betreated include eukaryotic cells, e.g., from a subject described above,or plant cells, yeast cells and prokaryotic cells, e.g., bacterialcells. For example, activating compounds may be administered to formanimals to improve their ability to withstand farming conditions longer.

Compounds may also be used to increase lifespan, stress resistance, andresistance to apoptosis in plants. In one embodiment, a compound isapplied to plants, either on a periodic basis or in fungi. In anotherembodiment, plants are genetically modified to produce a compound. Inanother embodiment, plants and fruits are treated with a compound priorto picking and shipping to increase resistance to damage duringshipping.

Compounds may also be used to increase lifespan, stress resistance andresistance to apoptosis in insects. In this embodiment, compounds wouldbe applied to useful insects, e.g., bees and other insects that areinvolved in pollination of plants. In a specific embodiment, a compoundwould be applied to bees involved in the production of honey. Generally,the methods described herein may be applied to any organism, e.g.,eukaryote, that may have commercial importance. For example, they can beapplied to fish (aquaculture) and birds (e.g., chicken and fowl).

Higher doses of compounds may also be used as a pesticide by interferingwith the regulation of silenced genes and the regulation of apoptosisduring development. In this embodiment, a compound may be applied toplants using a method known in the art that ensures the compound isbio-available to insect larvae, and not to plants.

Activated sirtuin proteins that are in vitro outside of a cell may beused, e.g., for deacetylating target proteins, thereby, e.g., activatingthe target proteins. Activated sirtuins may be used, e.g., for theidentification, in vitro, of previously unknown targets of sirtuindeacetylation, for example using 2D electrophoresis of acetyl labeledproteins.

At least in view of the link between reproduction and longevity (Longoand Finch, Science, 2002), the compounds can be applied to affect thereproduction of organisms such as insects, animals and microorganisms.

Inhibitory compounds may be used for similar purposes as highconcentrations of activating compounds can be used for. For example,inhibitory compounds may be used to stimulate acetylation of substratessuch as p53 and thereby increase apoptosis, as well as to reduce thelifespan of cells and organisms and/or rendering them more sensitive tostress.

Pharmaceutical Compositions and Methods

Pharmaceutical compositions for use in accordance with the presentmethods may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, activatingcompounds and their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration. In one embodiment, the compound isadministered locally, at the site where the target cells, e.g., diseasedcells, are present, i.e., in the blood or in a joint.

Compounds can be formulated for a variety of loads of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. For systemic administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneous. For injection, the compounds can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, thecompounds may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets, lozanges, or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

For administration by inhalation, the compounds may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin, for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Pharmaceutical compositions (including cosmetic preparations) maycomprise from about 0.00001 to 100% such as from 0.001 to 10% or from0.1% to 5% by weight of one or more compounds described herein.

In one embodiment, a compound described herein, is incorporated into atopical formulation containing a topical carrier that is generallysuited to topical drug administration and comprising any such materialknown in the art. The topical carrier may be selected so as to providethe composition in the desired form, e.g., as an ointment, lotion,cream, microemulsion, gel, oil, solution, or the like, and may becomprised of a material of either naturally occurring or syntheticorigin. It is preferable that the selected carrier not adversely affectthe active agent or other components of the topical formulation.Examples of suitable topical carriers for use herein include water,alcohols and other nontoxic organic solvents, glycerin, mineral oil,silicone, petroleum jelly, lanolin, fatty acids, vegetable oils,parabens, waxes, and the like.

Formulations may be colorless, odorless ointments, lotions, creams,microemulsions and gels.

Compounds may be incorporated into ointments, which generally aresemisolid preparations which are typically based on petrolatum or otherpetroleum derivatives. The specific ointment base to be used, as will beappreciated by those skilled in the art, is one that will provide foroptimum drug delivery, and, preferably, will provide for other desiredcharacteristics as well, e.g., emolliency or the like. As with othercarriers or vehicles, an ointment base should be inert, stable,nonirritating and nonsensitizing. As explained in Remington's, cited inthe preceding section, ointment bases may be grouped in four classes:oleaginous bases; emulsifiable bases; emulsion bases; and water-solublebases. Oleaginous ointment bases include, for example, vegetable oils,fats obtained from animals, and semisolid hydrocarbons obtained frompetroleum. Emulsifiable ointment bases, also known as absorbent ointmentbases, contain little or no water and include, for example,hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.Emulsion ointment bases are either water-in-oil (W/O) emulsions oroil-in-water (O/W) emulsions, and include, for example, cetyl alcohol,glyceryl monostearate, lanolin and stearic acid. Exemplary water-solubleointment bases are prepared from polyethylene glycols (PEGs) of varyingmolecular weight; again, reference may be had to Remington's, supra, forfurther information.

Compounds may be incorporated into lotions, which generally arepreparations to be applied to the skin surface without friction, and aretypically liquid or semiliquid preparations in which solid particles,including the active agent, are present in a water or alcohol base.Lotions are usually suspensions of solids, and may comprise a liquidoily emulsion of the oil-in-water type. Lotions are preferredformulations for treating large body areas, because of the ease ofapplying a more fluid composition. It is generally necessary that theinsoluble matter in a lotion be finely divided. Lotions will typicallycontain suspending agents to produce better dispersions as well ascompounds useful for localizing and holding the active agent in contactwith the skin, e.g., methylcellulose, sodium carboxymethylcellulose, orthe like. An exemplary lotion formulation for use in conjunction withthe present method contains propylene glycol mixed with a hydrophilicpetrolatum such as that which may be obtained under the trademarkAquaphor® from Beiersdorf, Inc. (Norwalk, Conn.).

Compounds may be incorporated into creams, which generally are viscousliquid or semisolid emulsions, either oil-in-water or water-in-oil.Cream bases are water-washable, and contain an oil phase, an emulsifierand an aqueous phase. The oil phase is generally comprised of petrolatumand a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phaseusually, although not necessarily, exceeds the oil phase in volume, andgenerally contains a humectant. The emulsifier in a cream formulation,as explained in Remington's, supra, is generally a nonionic, anionic,cationic or amphoteric surfactant.

Compounds may be incorporated into microemulsions, which generally arethermodynamically stable, isotropically clear dispersions of twoimmiscible liquids, such as oil and water, stabilized by an interfacialfilm of surfactant molecules (Encyclopedia of Pharmaceutical Technology(New York: Marcel Dekker, 1992), volume 9). For the preparation ofmicroemulsions, surfactant (emulsifier), co-surfactant (co-emulsifier),an oil phase and a water phase are necessary. Suitable surfactantsinclude any surfactants that are useful in the preparation of emulsions,e.g., emulsifiers that are typically used in the preparation of creams.The co-surfactant (or “co-emulsifer”) is generally selected from thegroup of polyglycerol derivatives, glycerol derivatives and fattyalcohols. Preferred emulsifier/co-emulsifier combinations are generallyalthough not necessarily selected from the group consisting of: glycerylmonostearate and polyoxyethylene stearate; polyethylene glycol andethylene glycol palmitostearate; and caprilic and capric triglyceridesand oleoyl macrogolglycerides. The water phase includes not only waterbut also, typically, buffers, glucose, propylene glycol, polyethyleneglycols, preferably lower molecular weight polyethylene glycols (e.g.,PEG 300 and PEG 400), and/or glycerol, and the like, while the oil phasewill generally comprise, for example, fatty acid esters, modifiedvegetable oils, silicone oils, mixtures of mono- di- and triglycerides,mono- and di-esters of PEG (e.g., oleoyl macrogol glycerides), etc.

Compounds may be incorporated into gel formulations, which generally aresemisolid systems consisting of either suspensions made up of smallinorganic particles (two-phase systems) or large organic moleculesdistributed substantially uniformly throughout a carrier liquid (singlephase gels). Single phase gels can be made, for example, by combiningthe active agent, a carrier liquid and a suitable gelling agent such astragacanth (at 2 to 5%), sodium alginate (at 2-10%), gelatin (at 2-15%),methylcellulose (at 3-5%), sodium carboxymethylcellulose (at 2-5%),carbomer (at 0.3-5%) or polyvinyl alcohol (at 10-20%) together andmixing until a characteristic semisolid product is produced. Othersuitable gelling agents include methylhydroxycellulose,polyoxyethylene-polyoxypropylene, hydroxyethylcellulose and gelatin.Although gels commonly employ aqueous carrier liquid, alcohols and oilscan be used as the carrier liquid as well.

Various additives, known to those skilled in the art, may be included informulations, e.g., topical formulations. Examples of additives include,but are not limited to, solubilizers, skin permeation enhancers,opacifiers, preservatives (e.g., anti-oxidants), gelling agents,buffering agents, surfactants (particularly nonionic and amphotericsurfactants), emulsifiers, emollients, thickening agents, stabilizers,humectants, colorants, fragrance, and the like. Inclusion ofsolubilizers and/or skin permeation enhancers is particularly preferred,along with emulsifiers, emollients and preservatives. An optimum topicalformulation comprises approximately: 2 wt. % to 60 wt. %, preferably 2wt. % to 50 wt. %, solubilizer and/or skin permeation enhancer; 2 wt. %to 50 wt. %, preferably 2 wt. % to 20 wt. %, emulsifiers; 2 wt. % to 20wt. % emollient; and 0.01 to 0.2 wt. % preservative, with the activeagent and carrier (e.g., water) making of the remainder of theformulation.

A skin permeation enhancer serves to facilitate passage of therapeuticlevels of active agent to pass through a reasonably sized area ofunbroken skin. Suitable enhancers are well known in the art and include,for example: lower alkanols such as methanol ethanol and 2-propanol;alkyl methyl sulfoxides such as dimethylsulfoxide (DMSO),decylmethylsulfoxide (C.sub.10 MSO) and tetradecylmethyl sulfboxide;pyrrolidones such as 2-pyrrolidone, N-methyl-2-pyrrolidone andN-(-hydroxyethyl)pyrrolidone; urea; N,N-diethyl-m-toluamide; C.sub.2-C.sub.6 alkanediols; miscellaneous solvents such as dimethyl formamide(DMF), N,N-dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol; andthe 1-substituted azacycloheptan-2-ones, particularly1-n-dodecylcyclazacycloheptan-2-one (laurocapram; available under thetrademark Azone® from Whitby Research Incorporated, Richmond, Va.).

Examples of solubilizers include, but are not limited to, the following:hydrophilic ethers such as diethylene glycol monoethyl ether(ethoxydiglycol, available commercially as Transcutol®) and diethyleneglycol monoethyl ether oleate (available commercially as Softcutol®);polyethylene castor oil derivatives such as polyoxy 35 castor oil,polyoxy 40 hydrogenated castor oil, etc.; polyethylene glycol,particularly lower molecular weight polyethylene glycols such as PEG 300and PEG 400, and polyethylene glycol derivatives such as PEG-8caprylic/capric glycerides (available commercially as Labrasol®); alkylmethyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone andN-methyl-2-pyrrolidone; and DMA. Many solubilizers can also act asabsorption enhancers. A single solubilizer may be incorporated into theformulation, or a mixture of solubilizers may be incorporated therein.

Suitable emulsifiers and co-emulsifiers include, without limitation,those emulsifiers and co-emulsifiers described with respect tomicroemulsion formulations. Emollients include, for example, propyleneglycol, glycerol, isopropyl myristate, polypropylene glycol-2 (PPG-2)myristyl ether propionate, and the like.

Other active agents may also be included in formulations, e.g., otheranti-inflammatory agents, analgesics, antimicrobial agents, antifungalagents, antibiotics, vitamins, antioxidants, and sunblock agentscommonly found in sunscreen formulations including, but not limited to,anthranilates, benzophenones (particularly benzophenone-3), camphorderivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoylmethanes (e.g., butyl methoxydibenzoyl methane), p-aminobenzoic acid(PABA) and derivatives thereof, and salicylates (e.g., octylsalicylate).

In certain topical formulations, the active agent is present in anamount in the range of approximately 0.25 wt. % to 75 wt. % of theformulation, preferably in the range of approximately 0.25 wt. % to 30wt. % of the formulation, more preferably in the range of approximately0.5 wt. % to 15 wt. % of the formulation, and most preferably in therange of approximately 1.0 wt. % to 10 wt. % of the formulation.

Topical skin treatment compositions can be packaged in a suitablecontainer to suit its viscosity and intended use by the consumer. Forexample, a lotion or cream can be packaged in a bottle or a roll-ballapplicator, or a propellant-driven aerosol device or a container fittedwith a pump suitable for finger operation. When the composition is acream, it can simply be stored in a non-deformable bottle or squeezecontainer, such as a tube or a lidded jar. The composition may also beincluded in capsules such as those described in U.S. Pat. No. 5,063,507.Accordingly, also provided are closed containers containing acosmetically acceptable composition as herein defined.

In an alternative embodiment, a pharmaceutical formulation is providedfor oral or parenteral administration, in which case the formulation maycomprises an activating compound-containing microemulsion as describedabove, but may contain alternative pharmaceutically acceptable carriers,vehicles, additives, etc. particularly suited to oral or parenteral drugadministration. Alternatively, an activating compound-containingmicroemulsion may be administered orally or parenterally substantiallyas described above, without modification.

Compounds described herein may be stored in oxygen free environmentaccording to methods in the art. For example, resveratrol or analogthereof can be prepared in an airtight capusule for oral administration,such as Capsugel from Pfizer, Inc.

Cells, e.g., treated ex vivo with a compound described herein, can beadministered according to methods for administering a graft to asubject, which may be accompanied, e.g., by administration of animmunosuppressant drug, e.g., cyclosporin A. For general principles inmedicinal formulation, the reader is referred to Cell Therapy: Stem CellTransplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn& W. Sheridan eds, Cambridge University Press, 1996; and HematopoieticStem Cell Therapy, E. D. Ball, J. Lister & P. Law, ChurchillLivingstone, 2000.

Kits

Also provided herein are kits, e.g., kits for therapeutic purposes orkits for modulating the lifespan of cells or modulating apoptosis. A kitmay comprise one or more activating or inhibitory compounds describedherein, and optionally devices for contacting cells with the compounds.Devices include syringes, stents and other devices for introducing acompound into a subject or applying it to the skin of a subject.

The present description is further illustrated by the followingexamples, which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication) are hereby expressly incorporated by reference.

The practice of the present methods will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES Example 1 Small Molecule Activators of SIRT1

To identify compounds that modulate SIRT1 activity, we screened a numberof small molecule libraries using a fluorescent deacetylation assay in96-well plates (Bitterman et al. J Biol Chem 277, 45099-107 (2002)). Thesubstrate used in the assay was a fluorogenic peptide based on thesequence encompassing the p53-K382 acetylation site, a known target ofSIRT1 in vivo (Vaziri et al. Cell 107, 149-59 (2001); Luo et al. Cell107, 137-48 (2001); Langley et al. EMBO J 21, 2383-2396 (2002)). Thissubstrate was preferred over a variety of other fluorogenic peptidesubstrates that were based on other known HDAC targets (FIG. 5). Thesmall molecule libraries included analogues of nicotinamide, ε-acetyllysine, NAD⁺, nucleotides, nucleotide analogues and purinergic ligands.From the initial screen, several sirtuin inhibitors were found(Supplementary Table 7). However, the most striking outcome was theidentification of two compounds, quercetin and piceatannol, thatstimulated SIRT1 activity five and eight-fold, respectively (Table 1).Both quercetin and piceatannol have been previously identified asprotein kinase inhibitors (Glossmann et al. Naunyn Schmiedebergs ArchPharmacol 317, 100-2 (1981); Oliver et al. J Biol Chem 269, 29697-703(1994)).

Comparison of the structures of the two activating compounds suggested apossible structure-activity relationship. Piceatannol comprises twophenyl groups trans to one another across a linking ethylene moiety. Thetrans-stilbene ring structures of piceatannol are superimposable on theflavonoid A and B rings of quercetin, with the ether oxygen and carbon-2of the C ring aligning with the ethylene carbons in piceatannol (seestructures, Table 1). Further, the 5,7,3′ and 4′ hydroxyl grouppositions in quercetin can be aligned, respectively, with the 3,5,3′ and4′ hydroxyls of piceatannol.

Given the demonstrated longevity-enhancing effects of sirtuin activityin S. cerevisiae( Kaeberlein et al. Genes Dev 13, 2570-80 (1999)) and C.elegans(Tissenbaum, H. A. and Guarente, L. Nature 410, 227-30. (2001)),it was naturally of interest to further explore the structure-activityrelationship among compounds that stimulate SIRT1. Both quercetin andpiceatannol are polyphenols, members of a large and diverse group ofplant secondary metabolites that includes flavones, stilbenes,flavanones, isoflavones, catechins (flavan-3-ols), chalcones, tanninsand anthocyanidins (Ferguson, L. R. Mutat Res 475, 89-111 (2001);Middleton et al. Pharmacol Rev 52, 673-751 (2000)). Polyphenolsnoteworthy with respect to potential longevity-enhancing effects includeresveratrol, a stilbene found in red wine and epigallocatechin gallate(EGCG) from green tea. Both have been suggested on the basis ofepidemiological and mechanistic investigations to exert cancerchemopreventive and cardioprotective effects (Ferguson, L. R. Mutat Res475, 89-111 (2001); Middleton et al. Pharmacol Rev 52, 673-751 (2000);and Jang et al. Science 275, 218-20 (1997)). We therefore performed asecondary screen encompassing resveratrol, EGCG and additionalrepresentatives from a number of the polyphenol classes listed above.The screen emphasized flavones due to the great number of hydroxylposition variants available in this group (Middleton et al. PharmacolRev 52, 673-751 (2000). The results of this screen are summarized inSupplementary Tables 1-6. In the tables, a “ratio to control rate” above1 indicates that a compound with such a rate is an activator of thesirtuin tested and a number under 1 indicates that a compound is aninhibitor.

Additional potent SIRT1 activators were found among the stilbenes,chalcones and flavones (Table 1, Supplementary Tables 1 and 2). The sixmost active flavones had 3′ and 4′ hydroxyls (Supplementary Table 2),although it should be noted that the most active compound overall,resveratrol (3,5,4′-trihydroxystilbene), was more active thanpiceatannol, which differs only by its additional 3′-hydroxyl (Table 1).The importance of the 4′-hydroxyl to activity is underscored by the factthat each of the 12 most stimulatory flavones share this feature(Supplementary Tables 1 and 2).

Many, but not all of the most active compounds include hydroxyls in thetwo meta positions (e.g. 5,7-dihydroxylated flavones) of the ring (Aring), trans to that with the 4′ or 3′,4′ pattern (B ring, see Table 1,Supplementary Tables 1 and 2). A potentially coplanar orientation of thetrans phenyl rings may be important for activity since catechins andflavanones, which lack the 2,3 double-bond, have weak activity despitehaving equivalent hydroxylation patterns to various stimulatory flavones(compare Supplementary Tables 2 and 3 with 4 and 5). The absence ofactivity in the isoflavone genistein, although hydroxylated in anequivalent way to the stimulatory compounds apigenin and resveratrol(see Supplementary Tables 1, 2 and 4), is consistent with the idea thatthe trans positioning and spacing of the hydroxylated rings contributesstrongly to activity.

The biological effects of polyphenols are frequently attributed toantioxidant, metal ion chelating and/or free-radical scavenging activity(Ferguson, L. R. Mutat Res 475, 89-111 (2001); Jang et al. Science 275,218-20 (1997)). We considered the possibility that the apparentpolyphenol stimulation of SIRT1 might simply represent the repair ofoxidative and/or metal-ion induced damage incurred during preparation ofthe recombinant protein. Two features of our results argue against thisbeing the case. First, a variety of free-radical protective compounds,including antioxidants, chelators and radical scavengers, failed tostimulate SIRT1 (see Supplementary Table 6.). Second, among variouspolyphenols of equivalent antioxidant capacity we observed diverse SIRT1stimulating activity (e.g. compare resveratrol, quercetin and theepicatechins in Supplementary Tables 1, 2 and 5 and see Stojanovic etal. Arch Biochem Biophys 391, 79-89 (2001)).

Example 2 Resveratrol's Effects on SIRT1 Kinetics

Detailed enzyme kinetic investigations were performed using the mostpotent activator, resveratrol. Dose-response experiments performed underthe conditions of the polyphenol screening assays (25 μM NAD⁺, 25 μMp53-382 acetylated peptide), showed that the activating effect doubledthe rate at ˜11 μM and was essentially saturated at 100 μM resveratrol(FIG. 1 a). Initial enzyme rates, in the presence or absence of 100 μMresveratrol, were determined either as a function of acetyl-peptideconcentration with high NAD⁺ (3 mM NAD⁺, FIG. 1 b) or as a function ofNAD⁺ concentration with high acetyl-peptide (1 mM p53-382 acetylatedpeptide, FIG. 1 c). Although resveratrol had no significant effect onthe two V_(max) determinations (FIGS. 1 b, 1 c), it had pronouncedeffects on the two apparent K_(m)s. Its effect on the acetylated peptideK_(m) was particularly striking, amounting to a 35-fold decrease (FIG. 1b). Resveratrol also lowered the K_(m) for NAD⁺ over 5-fold (FIG. 1 c).Since resveratrol acts only on K_(m), it could be classified as anallosteric effector of ‘K system’ type (Monod et al. J. Mol. Biol. 12,88-118 (1965)). This can imply that only the substrate binding affinityof the enzyme has been altered, rather than a rate-limiting catalyticstep.

Our previous kinetic analysis of SIRT1 and Sir2 (Bitterman et al. J BiolChem 277, 45099-107 (2002)) and our genetic analysis of Sir2's role inyeast lifespan extension (Anderson et al. Nature 423, 181-5 (2003);Anderson et al. J Biol Chem 277, 18881-90. (2002)) have implicatednicotinamide (a product of the sirtuin reaction) as a physiologicallyimportant inhibitor of sirtuin activity. Therefore the effects ofresveratrol on nicotinamide inhibition were tested. In experimentssimilar to those of FIGS. 1 b and 1 c, kinetic constants in the presenceof 50 μM nicotinamide were determined either by varying theconcentration of NAD⁺ or that of the p53-382 acetylated peptide (FIG. 1d). Nicotinamide, in contrast to resveratrol, affects the SIRT1 V_(max)(note 30% and 36% V_(max) decreases in absence of resveratrol, FIG. 1 dand see Bitterman et al. J Biol Chem 277, 45099-107 (2002)). In thepresence of 50 μM nicotinamide, resveratrol appears to have complex,concentration-dependent effects on the kinetics of SIRT1 (FIG. 1 d).Apparent K_(m) for NAD⁺ and acetylated substrate appear to actually beraised by 5 μM resveratrol when nicotinamide is present. At 20 and 100μM, in the presence of 50 μM nicotinamide, resveratrol lowers the K_(m)for both NAD⁺ and acetylated peptide, without reversing thenicotinamide-induced V_(max) decrease. It has been proposed thatsirtuins may bind nicotinamide at a second site, known as “the Cpocket”, distinct from the “B” site that interacts with the nicotinamidemoiety of NAD⁺ (Bitterman et al. J Biol Chem 277, 45099-107 (2002)). Inlight of this potential complexity, further kinetic studies,supplemented by structural/crystallographic information, will likely benecessary to fully elucidate the interplay between the effects ofnicotinamide and polyphenols.

Example 3 Activating Compounds Extend Yeast Lifespan

To investigate whether these compounds could stimulate sirtuins in vivo,we utilized S. cerevisiae, an organism in which the upstream regulatorsand downstream targets of Sir2 are relatively well understood. Aresveratrol dose-response study of Sir2 deacetylation rates (FIG. 2 a)indeed reveals that resveratrol stimulates Sir2 in vitro, with theoptimum concentration of activator being 2-5 μM. Levels of activationwere somewhat lower than those for SIRT1, and unlike SIRT1, inhibitionwas seen at concentrations greater than ˜100 μM.

Resveratrol and four other potent sirtuin activators, representatives ofthe stilbene, flavone, and chalcone families, were tested for theireffect on yeast lifespan. Due to the potential impediment by the yeastcell wall or plasma membrane and suspected slow oxidation of thecompound in the medium, we chose to use a concentration (10 μM) slightlyhigher than the optimal resveratrol concentration in vitro. As shown inFIG. 2 b, quercetin and piceatannol had no significant effect onlifespan. In contrast, butein, fisetin and resveratrol increased averagelifespan by 31, 55 and 70%, respectively, and all three significantlyincreased maximum lifespan (FIG. 2 c). Concentrations of resveratrolhigher than 10 μM provided no added lifespan benefit and there was nolasting effect of the compound on the lifespan of pre-treated youngcells (FIG. 2 d).

For subsequent yeast genetic experiments we focused on resveratrolbecause it was the most potent SIRT1 activator and provided the greatestlifespan extension. Glucose restriction, a form of CR in yeast, resultedin no significant extension of the long-lived resveratrol-treated cells(FIG. 3 a), indicating that resveratrol likely acts via the same pathwayas CR. Consistent with this, resveratrol had no effect on the lifespanof a sir2 null mutant (FIG. 3 b). Given that resveratrol is reported tohave fungicidal properties at high concentrations (Pont, V. and Pezet,R. J Phytopathol 130, 1-8 (1990)), and that mild stress can extend yeastlifespan by activating PNC1 (Anderson et al. Nature 423, 181-5 (2003)),it was plausible that resveratrol was extending lifespan by inducingPNC1, rather than acting on Sir2 directly. However, resveratrol extendedthe lifespan of a pnc1 null mutant nearly as well as it did wild typecells (FIG. 3 b). Together these data show that resveratrol actsdownstream of PNC1 and requires SIR2 for its effect. Thus, the simplestexplanation for our observations is that resveratrol increases lifespanby directly stimulating Sir2 activity.

A major cause of yeast aging is thought to stem from the inherentinstability of the repetitive rDNA locus (Sinclair, D. A. Mech AgeingDev 123, 857-67 (2002); Lin et al. Science 289, 2126-8 (2000); Sinclair,D. A. and Guarente, L. Cell 91, 1033-42 (1997); Defossez et al. Mol Cell3, 447-55 (1999); Park et al. Mol Cell Biol 19, 3848-56 (1999)).Homologous recombination between rDNA repeats can generate anextrachromosomal circular form of rDNA (ERC) that is replicated until itreaches toxic levels in old cells. Sir2 is thought to extend lifespan bysuppressing recombination at the replication fork barrier of rDNA(Benguria et al. Nucleic Acids Res 31, 893-8 (2003)). Consistent with thelifespan extension we observed for resveratrol, this compound reducedthe frequency of rDNA recombination by ˜60% (FIG. 3 c), in aSIR2-dependent manner (FIG. 3 d). In the presence of the Sir2 inhibitornicotinamide, recombination was also decreased by resveratrol (FIG. 3c), in agreement with the kinetic data (see FIG. 1 d). Interestingly, wefound that resveratrol and other sirtuin activators had only minoreffects on rDNA silencing (FIG. 3 e and f).

Another measure of lifespan in S. cerevisiae is the length of time cellscan survive in a metabolically active but nutrient deprived state. Agingunder these conditions (i.e. chronological aging) is primarily due tooxidative damage (Longo, V. D. and Finch, C. E. Science 299, 1342-6(2003)). Resveratrol (10 μM or 100 μM) failed to extend chronologicallifespan (not shown), indicating that the sirtuin-stimulatory effect ofresveratrol may be more relevant in vivo than its antioxidant activity(Ferguson, L. R. Mutat Res 475, 89-111 (2001); Middleton et al.Pharmacol Rev 52, 673-751 (2000)).

Example 4 Effects of Activators in Human Cells

To test whether these compounds could stimulate human SIRT1 in vivo, wefirst employed a cellular deacetylase assay that we had developed. Aschematic of the assay procedure is depicted in FIG. 4 a. Cells areincubated with media containing the fluorogenic ε-acetyl-lysinesubstrate, ‘Fluor de Lys’ (FdL). This substrate, neutral whenacetylated, becomes positively charged upon deacetylation andaccumulates within cells (see FIG. 6 a). Lysis of the cells and additionof the non-cell-permeable ‘Developer’ reagent releases a fluorophorspecifically from those substrate molecules that have been deacetylated(FIG. 4 a and see Methods). With HeLa cells growing adherently, 5-10% ofthe signal produced in this assay is insensitive to 1 μM trichostatin A(TSA), a potent inhibitor of class I and II HDACs but not sirtuins(class III) (Denu, J. M. Trends Biochem Sci 28, 41-8 (2003)). (FIGS. 6 band 6 c).

A selection of SIRT1-stimulatory and non-stimulatory polyphenols weretested for their effects on this TSA-insensitive signal (FIG. 4 b).Cellular deacetylation signals in the presence of each compound (y-axis,FIG. 4 b) were plotted against their fold-stimulations of SIRT1 in vitro(x-axis, FIG. 4 b, data from Supplementary Tables 1-3). For most of thecompounds, the in vitro activity roughly corresponded to the cellularsignal. Compounds with little or no in vitro activity clustered aroundthe negative control (Group A, FIG. 4 b). Another grouping, of strong invitro activators is clearly distanced from the low activity cluster inboth dimensions (Group B, FIG. 4 b). A notable outlier was butein, apotent activator of SIRT1 in vitro which had no effect on the cellularsignal. With allowances for possible variation among these compounds inproperties unrelated to direct sirtuin stimulation, such ascell-permeability and rates of metabolism, these data are consistentwith the idea that certain polyphenols can activate native sirtuins invivo.

One known target of SIRT1 in vivo is lysine 382 of p53. Deacetylation ofthis residue by SIRT1 decreases the activity and half-life of p53(Vaziri et al. Cell 107, 149-59 (2001); Luo et al. Cell 107, 137-48.(2001); Langley et al. EMBO J 21, 2383-2396 (2002)). To follow theacetylation status of K382 we generated a rabbit polyclonal antibodythat recognizes the acetylated form of K382 (Ac-K382) on Western blotsof whole cell lysates. As a control we showed that the signal wasspecifically detected in extracts from cells exposed to ionizingradiation (FIG. 4 c), but not in extracts from cells lacking p53 orwhere arginine had been substituted for lysine 382 (data not shown).U2OS osteosarcoma cells were pre-treated for 4 hours with resveratrol(0.5 and 50 μM) and exposed to UV radiation. We consistently observed amarked decrease in the level of Ac-K382 in the presence of 0.5 μMresveratrol, compared to untreated cells (FIG. 4 d). At higherconcentrations of resveratrol (>50 μM) the effect was reversed (FIG. 4 dand data not shown), consistent with previous reports of increased p53activity at such concentrations (Dong, Z. Mutat Res 523-524, 145-50(2003)). The ability of low concentrations of resveratrol to promotedeacetylation of p53 was diminished in cells expressing adominant-negative SIRT1 allele (H363Y) (FIG. 4 e), demonstrating thatSIRT1 is necessary for this effect. This biphasic dose-response ofresveratrol could explain the dichotomy in the literature regarding theeffects of resveratrol on cell survival (Ferguson, L. R. Mutat Res 475,89-111 (2001); Dong, Z. Mutat Res 523-524, 145-50 (2003); Nicolini etal. Neurosci Lett 302, 41-4 (2001)).

Thus, we have discovered the first known class of small molecule sirtuinactivators, all of which are plant polyphenols. These compounds candramatically stimulate sirtuin activity in vitro and promote effectsconsistent with increased sirtuin activity in vivo. In human cells,resveratrol promotes SIRT1-mediated p53 deacetylation of K382. In yeast,the effect of resveratrol on lifespan is as great as anylongevity-promoting genetic manipulation (Anderson et al. Nature 423,181-5 (2003)) and has been linked convincingly to the direct activationof Sir2. The correlation between lifespan and rDNA recombination, butnot silencing, adds to the body of evidence that yeast aging is due toDNA instability (Sinclair, D. A. Mech Ageing Dev 123, 857-67 (2002); Linet al. Science 289, 2126-8 (2000); Sinclair, D. A. and Guarente, L. Cell91, 1033-42. (1997); Defossez et al. Mol Cell 3, 447-55 (1999); Park etal. Mol Cell Biol 19, 3848-56 (1999)) not gene dysregulation (Jazwinski,S. M. Ann N Y Acad Sci 908, 21-30 (2000)).

Sirtuins have been found in diverse eukaryotes, including fungi,protozoans, metazoans and plants (Pandey et al. Nucleic Acids Res 30,5036-55 (2002); Frye, R. A. Biochem Biophys Res Commun 273, 793-8(2000)), and likely evolved early in life's history (Kenyon, C. Aconserved regulatory mechanism for aging. Cell 105, 165-168 (2001)).Plants are known to produce a variety of polyphenols, includingresveratrol, in response to stresses such as dehydration, nutrientdeprivation, UV radiation and pathogens (Soleas et al. Clin Biochem 30,91-113 (1997); Coronado et al. Plant Physiol 108, 533-542 (1995)).Therefore it is believed that these compounds may be synthesized toregulate a sirtuin-mediated plant stress response. This would beconsistent with the recently discovered relationship betweenenvironmental stress and Sir2 activity in yeast (Anderson et al. Nature423, 181-5 (2003)). Perhaps these compounds have stimulatory activity onsirtuins from fungi and animals because they mimic an endogenousactivator, as is the case for the opiates/endorphins,cannabinols/endocannabinoids and various polyphenols with estrogen-likeactivity (Ferguson, L. R. Mutat Res 475, 89-111 (2001); Middleton et al.Pharmacol Rev 52, 673-751 (2000)). Alternatively, animal and fungalsirtuins may have retained or developed an ability to respond to theseplant metabolites because they are a useful indicator of a deterioratingenvironment and/or food supply.

Example 5 Materials and Methods for Examples 1-4

Compound Libraries and Deacetylation Assays

His₆-tagged recombinant SIRT1 and GST-tagged recombinant Sir2 wereprepared as described by (Bitterman et al. J Biol Chem 277, 45099-107.(2002). From 0.1 to 1 μg of SIRT1 and 1.5 μg of Sir2 were used perdeacetylation assay (in 50 μl total reaction). SIRT1 assays and certainof those for Sir2 employed the p53-382 acetylated substrate (‘Fluor deLys-SIRT1′, BIOMOL) rather than FdL.

Themed compound libraries (BIOMOL) were used for primary and secondaryscreening. Most polyphenol compounds were dissolved at 10 mM indimethylsulfoxide (DMSO) on the day of the assay. For water solublecompounds and negative controls, 1% v/v DMSO was added to the assay. Invitro fluorescence assay results were read in white ½-volume 96-wellmicroplates (Corning Costar 3693) with a CytoFluor™II fluorescence platereader (PerSeptive Biosystems, Ex. 360 nm, Em. 460 nm, gain=85). HeLacells were grown and the cellular deacetylation assays were performedand read, as above, but in full-volume 96-well microplates (CorningCostar 3595). Unless otherwise indicated all initial rate measurementswere means of three or more replicates, obtained with single incubationtimes, at which point 5% or less of the substrate initially present hadbeen deacetylated. Calculation of net fluorescence increases includedsubtraction of a blank value, which in the case of Sir2 was obtained byomitting the enzyme from the reaction and in the case of SIRT1 by addingan inhibitor (200 μM suramin or 1 mM nicotinamide) to the reaction priorto the acetylated substrate. A number of the polyphenols partiallyquenched the fluorescence produced in the assay and correction factorswere obtained by determining the fluorescence increase due to a 3 μMspike of an FdL deacetylated standard (BIOMOL, catalog number KI-142).All error bars represent the standard error of the mean.

Media and Strains

All yeast strains were grown at 30° C. in complete yeastextract/bactopeptone, 2.0% (w/v) glucose (YPD) medium except wherestated otherwise. Calorie restriction was induced in 0.5% glucose.Synthetic complete (SC) medium consisted of 1.67% yeast nitrogen base,2% glucose, 40 mg/liter each of auxotrophic markers. SIR2 was integratedin extra copy and disrupted as described by Lin et al. (Science 289,2126-8 (2000)). Other strains are described elsewhere (Bitterman et al.J Biol Chem 277, 45099-107 (2002)). For cellular deacetylation assays,HeLa S3 cells were used. U2OS osteosarcoma and human embryonic kidney(HEK 293) cells were cultured adherently in Dulbecco's Modified Eagle'sMedium (DMEM) containing 10% fetal calf serum (FCS) with 1.0% glutamineand 1.0% penecillin/streptomycin. HEK 293 overexpressing dominantnegative SIRT1 H363Y was a gift of R. Frye (U. Pittsburgh).

Lifespan Determinations

Lifespan measurements were performed using PSY316AT MATα as previouslydescribed by Anderson et al. (J Biol Chem 277, 18881-90. (2002). Allcompounds for lifespan analyses were dissolved in 95% ethanol and plateswere dried and used within 24 hours. Prior to lifespan analysis, cellswere pre-incubated on their respective media for at least 15 hours.Following transfer to a new plate, cells were equilibrated on the mediumfor a minimum of 4 hours prior to micro-manipulating them. At least 30cells were examined per experiment and each experiment was performed atleast twice. Statistical significance of lifespan differences wasdetermined using the Wilcoxon rank sum test. Differences are stated tobe significant when the confidence is higher than 95%.

Silencing and Recombination Assays

Ribosomal DNA silencing assays using the URA3 reporters were performedas previously described by Bitterman et al. (J Biol Chem 277, 45099-107(2002)). Ribosomal DNA recombination frequencies were determined byplating W303AR cells (Sinclair, D. A. and Guarente, L. Cell 91, 1033-42(1997)) on YPD medium with low adenine/histidine and counting thefraction of half-red sectored colonies using Bio-Rad Quantity Onesoftware as described by Anderson et al. (J Biol Chem 277, 18881-90.(2002)). At least 6000 cells were analyzed per experiment and allexperiments were performed in triplicate. All strains were pre-grown for15 hours with the relevant compound prior to plating.

Proteins and Western Analyses

Recombinant Sir2-GST was expressed and purified from E. coli aspreviously described except that lysates were prepared using sonication(Bitterman et al. J Biol Chem 277, 45099-107 (2002). Recombinant SIRT1from E. coli was prepared as previously described (Bitterman et al. JBiol Chem 277, 45099-107 (2002). Polyclonal antiserum against p53-AcK382was generated using an acetylated peptide antigen as previouslydescribed (Vaziri et al. Cell 107, 149-59 (2001) with the followingmodifications. Anti-Ac-K382 antibody was affinity purified usingnon-acetylated p53-K382 peptides and stored in PBS at −70° C. andrecognized an acetylated but not a non-acetylated p53 peptide. Westernhybridizations using anti-acetylated K382 or anti-actin (Chemicon)antibody were performed at 1:1000 dilution of antibody. Hybridizationswith polyclonal p53 antibody (Santa Cruz Biotech.) used 1:500 dilutionof antibody. Whole cell extracts were prepared by lysing cells in buffercontaining 150 mM NaCl, 1 mM MgCl₂, 10% glycerol, 1% NP40, 1 mM DTT andanti-protease cocktail (Roche).

Example 6 Localization of the Activation Domain of Sirtuins to theirN-Terminus

Yeast Sir2 and human SIRT1 are very homologous and differ from humanSIRT2 by the addition of an N-terminal domain that is absent in SIRT2.The effect of resveratrol was assayed on human recombinant SIRT2 asfollows. Human recombinant SIRT2 was incubated at a concentration of1.25 μg/well with 25 μM of Fluor de Lys-SIRT2 (BIOMOL cat. # KI-179) and25 μM NAD⁺ for 20 minutes at 37° C., as described above. Results,indicate that, in contrast to SIRT1, increasing concentrations ofresveratrol decrease SIRT2 activity. Thus, based on the difference instructure of SIRT1 and SIRT2, i.e., the absence of an N-terminal domain,it is believed that the N-terminal domain of SIRT1 and Sir2 is necessaryfor activation by the compounds described herein. In particular, it islikely that the activator compounds described herein interact with theN-terminal portion of sirtuins. The N-terminal portion of SIRT1 that isnecessary for the action of the compounds is from about amino acid 1 toabout amino acid 176, and that of Sir2 is from about amino acid 1 toabout amino acid 175.

Example 7 Resveratrol Extends the Lifespan of C. elegans

50 C. elegans worms (strain N2) were grown in the presence or absence of100 μM resveratrol for 17 days. On day 17, only 5 worms in the controlgroup without resveratrol were alive, whereas 17 worms were alive in thegroup that was treated with resveratrol. Thus, the presence ofresveratrol in the growth media of C. elegans extends their lifespan.

Example 8 Identification of Additional Activators of Sirtuins

Using the screening assay described in Example 1, five more sirtuinactivators have been identified. These are set forth in supplementaryTable 8.

Example 9 Identification of Inhibitors of Sirtuins

Using the screening assay described in Example 1, more inhibitors wereidentified. These are set forth in the appended supplementary Table 8,and correspond to the compounds having a ratio to control rate of lessthan 1.

Example 10 Identification of Further Activators and Inhibitors ofSirtuins

Additional activators and inhibitors of sirtuins were identified, andare listed in Tables 9-13. In these Tables, “SE” stands for Standarderror of the mean and N is the number of replicates used to calculatemean ratio to the control rate and standard error.

All SIRT1 rate measurements used in the calculation of “Ratio to ControlRate” were obtained with 25 μM NAD⁺ and 25 μM p53-382 acetylated peptidesubstrate were performed as described above and in K. T. Howitz et al.Nature (2003) 425: 191. All ratio data were calculated from experimentsin which the total deacetylation in the control reaction was 0.25-1.25μM peptide or 1-5% of the initial concentration of acetylated peptide.

Stability determinations (t_(1/2)) derived from SIRT1 rate measurementsperformed in a similar way to those described above, except that 5 μMp53-382 acetylated peptide substrate was used rather than 25 μM. Thefold-stimulation (ratio to control) obtained with a compound dilutedfrom an aged stock solution was compared to an identical dilution from astock solution freshly prepared from the solid compound. “t_(1/2)” isdefined as the time required for the SIRT1 fold-stimulation of thecompound from the aged solution to decay to one-half of that obtainedfrom a freshly prepared solution. Ethanol stocks of resveratrol, BML-212and BML-221 were prepared at 2.5 mM and the compounds were assayed at afinal concentration of 50 μM. The water stock of resveratrol was 100 μMand the assay performed at 10 μM. Stocks were aged by storage at room 5temperature, in glass vials, under a nitrogen atmosphere.

The effect of some of these compounds on lifespan was determined inyeast, C. elegans and D. melanogaster, as described above. The resultsare set forth below in Table A: % change in yeast % change in C. elegans% change in D. replicative lifespan lifespan relative to melanogasterlifespan relative to untreated untreated organisms relative to untreatedCompound organisms (10 μM)^(a) (100/500 μM)^(b) (100 μM)^(c) untreated100% 100% 100% Resveratrol 170-180% 110% 130%3,5,4′-Trihydroxy-trans-stilbene (from M. Tatar) Pinosylvin 114% ? ?3,5-Dihydroxy-trans-stilbene BML-212  98% ? ?3,5-Dihydroxy-4′-fluoro-trans-stilbene BML-217  90% ? ?3,5-Dihydroxy-4′-chloro-trans-stilbene BML-221 165% >100% (ongoing) ?3,4′-Dihydroxy-5-acetoxy-trans-stilbene BML-233 ? 70% (10)  ?3,5-Dihydroxy-4′-methoxy-trans-stilbene 50% (500)^(a)Replicative lifespans performed using 2% (w/v) glucose standardyeast compete medium (YPD) under standard conditions.^(b)Lifespan assays performed on N2 worms using E. coli as food understandard conditions.^(c)Lifespan assays preformed using 1.5% yeast as food supply underotherwise standard conditions.

The results indicate that resveratrol significantly extends lifespan inyeast, C. elegans and in D. melanogaster. Since BML-233 was shown to bea strong activator of 15 sirtuins (see above), the results obtained inC. elegans may indicate that the compound is toxic to the cells.

Without wanting to be limited to particular structures, it appears thatthe following structure/activity relationships exist. SIRT1 activationresults from several of these new analogs confirmed the importance ofplanarity, or at least the potential for planarity, between and withinthe two rings of the active compounds. Reduction of the double bond ofthe ethylene function, between, the two rings essentially abolishesactivity (compare Resveratrol, Table A and Dihydroresveratrol, Table E).Replacement of a phenyl moiety with a cyclohexyl group is nearly asdetrimental to SIRT1 stimulating activity (compare Pinosylvin, Table 9and BML-224, Table 12). Amide bonds are thought to have a partiallydouble bond character. However, replacement of the ethylene functionwith a carboxamide abolished activity (compare Pinosylvin, Table 9, withBML-219, Table 13). It is possible that this effect could be due in partto the position that carbonyl oxygen must assume in the conformationthat places the two rings trans to one another. If so, a compound inwhich the positions of the amide nitrogen and carbonyl are reversedmight be expected to have greater activity.

In twelve of the analogs resveratrol's 4′-hydroxy was replaced withvarious functionalities (see Tables 9 and 10, BML-221 in Table 11,BML-222 in Table 12). Although none of the replacements tried led tosubstantial increases in SIRT1 stimulating activity, this parameter was,in general, remarkably tolerant of substitutions at this position. Smallgroups (H— in Pinosylvin, Cl— in BML-217, —CH₃ in BML-228) did the leastto decrease activity. There is some evidence of a preference in theenzyme's stilbene binding/activation site for unbranched (ethyl inBML-225, azido in BML-232, —SCH₃ in BML-230) and hydrophobic functions(compare isopropyl in BML-231 to acetoxy in BML-221, acetamide inBML-222). Solution stability relative to resveratrol was stronglyincreased by one of the two 4′-substitutions (acetoxy, BML-221) testedfor this so far.

Resveratrol is currently the most potent known activator of SIRT1. Thecollection of analogs described above, particularly the group entailingsubstitutions at the 4′ position, may be instrumental in informing thedesign of new SIRT1 ligands with improved pharmacological properties.One parameter that may be of interest in this regard is stability. One4′-substituted analog, BML-221, displays a vast improvement in solutionstability relative to resveratrol and although diminished in vitro SIRT1activating ability, retains much of resveratrol's biological activity(see lifespan data). The 4′-hydroxyl of resveratrol is thought to be ofprimary importance to resveratrol's free-radical scavenging reactivity(S. Stojanovic et al. Arch. Biochem. Biophys. 2001 391 79). Most of the4′-substituted analogs have yet to be tested for solution stability, butif resveratrol's instability in solution is due to redox reactivity,many of the other analogs would be expected to also exhibit improvedstability.

The results obtained with 4′-substituted analogs may indicate promisingroutes to explore while seeking to increase SIRT1 binding affinity. Forexample, the efficacy of the 4′-ethyl compound (BML-225) might indicatethe presence of a narrow, hydrophobic binding pocket at the SIRT1 sitecorresponding to the 4′ end of resveratrol. Several new series of4′-substituted analogs are planned, the simplest comprisingstraight-chain aliphatic groups of various lengths.

Example 11 Methods of Synthesis of the Compounds in Tables 9-13

Most of the resveratrol analogs were synthesized by the same generalprocedure, from a pair of intermediates, a benzylphosphonate and analdehyde. The synthesis or sources of these intermediates are describedin section II. Section III. describes the procedures for synthesizingthe final compounds from any of the benzylphosphonate/aldehyde pairs.The coupling reaction (Section III. A.) is followed by one of twodeprotection reactions depending on whether the intermediates containedmethoxymethyl (Section III. B.) or methoxy (Section III. C.) protectinggroups. Section IV corresponds to Tables 14-18, which list theparticular benzylphosphonate and aldehyde used in the synthesis ofparticular final compounds. Seven of the compounds—Resveratrol,3,5-Dihydroxy-4′-methoxy-trans-stilbene, Rhapontin aglycone, BML-227,BML-221, Dihydroresveratrol, BML-219—were not synthesized by the generalprocedure and “N/A” appears next to their entries in the table.Resveratrol was from BIOMOL and the syntheses of the remaining compoundsare described in Section V.

II. Synthetic Intermediates

A. Benzylphosphonates (Synthesized)

Synthesis of Diethyl 4-Acetamidobenzylphosphonate: To diethyl4-aminobenzylphosphonate in 1:1 methylene chloride/pyridine was addedcatalytic DMAP and acetic anhydride (1.1 eq.). After 3 hours, thereaction was evaporated to dryness and purified via flash chromatography(silica gel).

Synthesis of Diethyl 4-Methylthiobenzylphosphonate: 4-Methylthiobenzylchloride was heated with triethylphosphite (as solvent) at 120° C.overnight. Excess triethyl phosphite was distilled off under high vacuumand heat. Flash chromatography (silica gel) yielded the desired product.

Synthesis of Diethyl 3,5-Dimethoxybenzylphosphonate: From3-5-Dimethoxybenzyl bromide. See synthesis of Diethyl4-Methylthiobenzylphosphonate.

Synthesis of Diethyl 4-Fluorobenzylphosphonate: From4-Fluorobenzylphosphonate. See synthesis of Diethyl4-Methylthiobenzylphosphonate.

Synthesis of Diethyl 4-azidobenzylphosphonate: To diethyl4-aminobenzylphosphonate in acetonitrile (2.5 mL) at 0° C. was added 6MHCl (1 mL). Sodium nitrite (1.12 eq.) in water (1 mL) was added dropwise and the resulting solution stirred at 0° C. for 30 mins. Sodiumazide (8 eq.) in water (1 mL) added drop wise (bubbling) and thesolution stirred at 0° C. for 30 mins., then at room temperature for 1hour. The reaction was diluted with ethyl acetate and washed with waterand brine and dried over sodium sulfate. Flash chromatography (silicagel) yielded the desired product.

B. Aldehydes (Synthesized)

Synthesis of 3,5-Dimethoxymethoxybenzaldehyde: To3,5-dihydroxybenzaldehyde in DMF at 0° C. was added sodium hydride (2.2eq.). The reaction was stirred for 30 min. at 0° C. Chloromethylmethylether (2.2 eq.) was added neat, drop wise and the reaction allowed towarm to room temperature over 1.5 hrs. The reaction mixture was dilutedwith diethyl ether and washed with water (2×) and brine (1×) and driedover sodium sulfate. Flash chromatography (silica gel) yielded thedesired product.

C. Purchased Intermediates: Unless Listed Above, all SyntheticIntermediates were Purchase from Sigma-Aldrich.

III. General Procedure for the Synthesis of Resveratrol Analogues

A. Benzylphosphonate/Aldehyde Coupling Procedure

To the appropriate benzylphosphonate (1.2 eq.) in dimethylformamide(DMF) at room temperature was added sodium methoxide (1.2 eq.). Thissolution was allowed to stir at room temperature for approximately 45minutes. The appropriate aldehyde (1 eq.) was then added (neat or in asolution of dimethylformamide). The resulting solution was then allowedto stir overnight at room temperature. Thin layer chromatography (TLC)was used to determine completeness of the reaction. If the reaction wasnot complete, the solution was heated at 45-50° C. until complete. Thereaction mixture was poured into water and extracted with ethyl acetate(2×). The combined organic layers were washed with brine and dried oversodium sulfate. Flash chromatography (silica gel) yielded the desiredproducts.

B. General Procedure for the Deprotection of MethoxymethylresveratrolAnalogues

To the appropriate methoxymethylstilbene derivative in methanol wasadded two drops of concentrated HCl. The resulting solution was heatedovernight at 50° C. The solution was evaporated to dryness uponcompletion of the reaction. Flash chromatography (silica gel) yieldedthe desired product.

C. General Procedure for the Deprotection of MethoxyresveratrolAnalogues

To the appropriate methoxystilbene derivative in methylene chloride wasadded tetrabutylammonium iodide (1.95 eq. per methoxy group). Thereaction was cooled to 0° C. and boron trichloride (1 M in methylenechloride; 2 eq. per methoxy group) was added dropwise. Following theaddition of boron trichloride, the cooling bath was removed and thereaction allowed to stir at room temperature until complete (asindicated by TLC). Saturated sodium bicarbonate solution was added andthe reaction vigorously stirred for 1 hour. The reaction was poured intocold 1M HCl and extracted with ethyl acetate (3×). The combined organiclayers were washed with water (1×) and brine (1×) and dried over sodiumsulfate. Flash chromatography (silica gel) yielded the desired products.

V. Special Syntheses

Synthesis of BML-219 (N-(3,5-Dihydroxyphenyl)benzamide): To benzoylchloride (1 eq.) in dry methylene chloride at room temperature was addedtriethylamine (1.5 eq.) and a catalytic amount of DMAP followed by3,5-dimethoxyaniline (1 eq.). The reaction was allowed to stir overnightat room temperature. Upon completion, the reaction was diluted withethyl acetate and washed with 1M HCl, water and brine and dried oversodium sulfate. Flash chromatography (silica gel) yielded themethoxystilbene derivative. To the methoxystilbene in dry methylenechloride at 0° C. was added tetrabutylammonium iodide (3.95 eq.)followed by boron trichloride (4 eq.; 1M in methylene chloride). Uponcompletion of the reaction (TLC), saturated sodium bicarbonate was addedand the mixture was vigorously stirred for 1 hour. The reaction wasdiluted with ethyl acetate and washed with 1M HCl and brine and driedover sodium sulfate. Flash chromatography (silica gel) yielded thedesired product.

Synthesis of BML-220 (3,3′,5-trihydroxy-4′-methoxystilbene): ToRhapontin in methanol was added catalytic p-toluenesulfonic acid. Thereaction was refluxed overnight. Upon completion of the reaction (TLC),the reaction mixture was evaporated to dryness and taken up in ethylacetate. The organics were washed with water and brine and dried oversodium sulfate. Flash chromatography (silica gel) yielded the desiredproduct.

Synthesis of BML-233 (3,5-Dihydroxy-4′-methoxystilbene): Todeoxyrhapontin in methanol was added catalytic p-toluenesulfonic acid.The reaction was refluxed overnight. Upon completion of the reaction(TLC), the reaction mixture was evaporated to dryness and taken up inethyl acetate. The organics were washed with water and brine and driedover sodium sulfate. Flash chromatography (silica gel) yielded thedesired product.

Synthesis of BML-221 and 227 (4′ and 3 monoacetylresveratrols): Toresveratrol in tetrahydrofuran at room temperature was added pyridine (1eq.) followed by acetic anhydride (1 eq.). After stirring for 48 hrs.,another 0.25 eq. acetic anhydride added followed by 24 hrs. of stirring.The reaction was diluted with methylene chloride (reaction was notcomplete) and washed with cold 0.5M HCl, water and brine. Organics weredried over sodium sulfate. Flash chromatography yielded a mixture of 4′-and 3-acetyl resveratrols. Preparative HPLC yielded both monoacetylresveratrols.

Synthesis of Dihydroresveratrol: To resveratrol in argon-purged ethylacetate in a Parr shaker was added 10% palladium on carbon (10 wt %).The mixture was shaken under an atmosphere of hydrogen (30 psi) for 5hours. Filtration through a pad of celite yielded the desired material.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims. TABLE 1 APPENDIX OF TABLES:Stimulation of SIRT1 Catalytic Rate by Plant Polyphenols (100 μM). Ratioto Control Rate Compound Mean ± SE Structure Resveratrol(3,5,4′-Trihydroxy-trans-stilbene) 13.4 ± 1.0 

Butein (3,4,2′,4′-Tetrahydroxychalcone) 8.53 ± 0.89

Piceatannol (3,5,3′,4′-Tetrahydroxy-trans-stilbene) 7.90 ± 0.50

Isoliquiritigenin (4,2′,4′-Trihydroxychalcone) 7.57 ± 0.84

Fisetin (3,7,3′,4′-Tetrahydroxyflavone) 6.58 ± 0.69

Quercetin (3,5,7,3′,4′-Pentahydroxyflavone) 4.59 ± 0.47

Abbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All ratio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

Supplementary Table 1. Effects of Stilbenes and Chalcones (100 μM) onSIRT1 Rate. Ratio to Control Rate Compound Mean ± SE ReplicatesStructure Skeleton Resveratrol (3,5,4′-Trihydroxy- trans-stilbene)Piceatannol # (3,5,3,′4′- Tetrahydroxy-trans-stilbene) Deoxyrhapontin(3,5-Dihydroxy-4′-methoxystilbene 3-O-β-D-glucoside) trans-StilbeneRhapontin 3,3′,5-Trihydroxy-4′-methoxystilbene 3-O-β-D-glucosidecis-Stilbene 13.4 ± # 1.0   7.90 ± 0.50  1.94 ± 0.21   1.48 ± 0.15  1.40± 0.37−  1.14 ± 0.29 10   7  6   6 6   6

Butein (3,4,2′,4′- Tetrahydroxychalcone) 4,2′,4′- Trihydroxychalcone3,4,2′,4′,6′- Pentahydroxychalcone Chalcone 8.53 ± 0.89  7.57 ± 0.84 2.80 ± 0.32  1.34 ± 0.17 6  6  6  6

Abbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All radio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

Supplementary Table 2. Effects of Flavones (100 μM) on SIRT1 Rate (PartI). Ratio to Control Rate Compound Mean ± SE Replicates StructureSkeleton Fisetin (3,7,3′,4′- Tetrahydroxyflavone) 5,7,3′4′,5′-Pentahydroxyflavone Luteolin (5,7,3′,4′- Tetrahydroxyflavone) 3,6,3′,4′-Tetrahydroxyflavone Quercetin (3,5,7,3′,4′- Pentahydroxyflavone)7,3′,4′,5′- Tetrahydroxyflavone 6.58 ± # 0.69   6.05 ± 0.98  5.66 ± 0.80  5.45 ± 0.57  4.59 ± 0.47   3.62 ± 0.56 9   6  6   12  16   6

Kaempferol 3.55 ± 0.56 6 (3,5,7,4′- Tetrahydroxyflavone)6-Hydroxyapigenin 3.06 ± 0.29 6 (5,6,7,4′- Tetrahydroxyflavone;Scutellarein) Apigenin 2.77 ± 0.40 6 (5,7,4′- Trihydroxyflavone)3,6,2′,4′- 2.10 ± 0.22 6 Tetrahydroxyflavone 7,4′-Dihydroxyflavone 1.91± 0.17 6Abbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All ratio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

Supplementary Table 3. Effects of Flavones (100 μM) on SIRT1 Rate (PartII). Ratio to Control Rate Compound Mean ± SE Replicates StructureSkeleton 7,8,3,4′- Tetrahydroxyflavone 3,6,2′,3′- Tetrahydroxyflavone4′-Hydroxyflavone 5,4′-Dihydroxyflavone 5,7-Dihydroxyflavone Morin(3,5,7,2′,4′- Pentahydroxyflavone) Flavone 1.91 ± # 0.39  1.74 ± 0.27 1.73 ± 0.12 1.56 ± 0.15 1.51 ± 0.18 1.461 ± 0.071   1.41 ± 0.23 6  6  66 6 6   6

5-Hydroxyflavone 1.22 ± 0.19 6 Myricetin 0.898 ± 0.070 12 (Cannabiscetin; 3,5,7,3′,4′,5′- Hexahydroxyflavone) 3,7,3′,4′,5′- 0.826± 0.074 12  Pentahydroxyflavone Gossypetin 0.723 ± 0.062 6(3,5,7,8,3′,4′- Hexahydroxyflavone)Abbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All ratio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

Supplementary Table 4. Effects of isoflavones, Flavanones andAnthocyanidins (100 μM) on SIRT1 Rate Ratio to Control Rate CompoundMean ± SE Replicates Structure Skeleton Daidzein(7,4′-Dihydroxyisoflavone) (5,7,4′- Trihydroxyisoflavone) 2.28 ± 0.74 1.109 ± 0.026 2  2

Naringenin (5,7,4′- Trihydroxyflavanone) 3,5,7,3′,4′-Pentahydroxyflavanone Flavanone 2.10 ± 0.23   1.97 ± 0.22  1.92 ± 0.24 6  5  6

Pelargonidin chloride (3,5,7.4′- Tetrahydroxyflavylium chloride)Cyanidin chloride (3,5,7,3′,4′- Pentahydroxyflavylium chloride)Delphinidin chloride (3,5,7,3′,4′,5′- Hexahydroxyflavylium chloride)1.586 ± # 0.037    0.451 ± 0.015    0.4473 ± 0.0071 2    2    2

Abbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All ratio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

Supplementary Table 5. Effects of Catechins (Flavan-3-ols) (100 μM) onSIRT1 Rate. Ratio to Control Rate Compound Mean ± SE ReplicatesStructure Skeleton/Structure (−)-Epicatechin (Hydroxy Sites:3,5,7,3′,4′) (−)-Catechin (Hydroxy Sites: 3,5,7,3′,4′) (−)-Gallocatechin(Hydroxy Sites: 3,5,7,3′,4′,5′) (+)-Catechin (Hydroxy Sites:3,5,7,3′,4′) (+)-Epicatechin (Hydroxy Sites: 3,5,7,3′,4′)(−)-Epigallocatechin (Hydroxy Sites: 3,5,7,3′,4′,5′) 1.53 ± # 0.31  1.41± 0.21  1.35 ± 0.25  1.31 ± 0.19  1.26 ± 0.20  0.41 ± 0.11 4  4  4  4  4 4

(−)-Epigallocatechin Gallate (Hydroxy Sites: 3*,5,7,3′,4′,5′; *Positionof gallate ester) 0.32 ± 0.12 4

Abbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All ratio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

Supplementary Table 6. Effects of Free Radical Protective Compounds (100μM) on SIRT1 Rate. Ratio to Control Rate Protective Compound Mean ± SEReplicates Mechanism Hinokitiol 2.48 ± 0.15 2 Iron Chelatol(b-Thujaplicin; 2- hydroxy-4-isopropyl-2,4,6- cycloheptatrien-1-one)L-(+)-Ergothioneine 2.06 ± 0.48 2 Antioxidant, ((S)-a-Carboxy-2,3-Peroxynitrite dihydro-N,N,N-trimethyl-2- Scavenger thioxo-1H-imidazole-4-ethanaminium inner salt) Caffeic Acid Phenyl Ester 1.80 ± 0.16 2 IronChelator MCI-186 1.2513 ± 0.0080 2 Radical (3-Methyl-1-phenyl-2-Scavenger pyrazolin-5-one) and Antioxidant HBED 1.150 ± 0.090 2 IronChelator (N,N′-Di-(2-hydroxy- benzyl)ethylenediamine- N,N′-diaceticacid.HCl.H2O) Ambroxol  1.075 ± 0.0026 2 Radical (trans-4-(2-Amino-3,5-Scavenger dibromobenzylamino) cyclohexane.HCl) U-83836E 1.030 ± 0.055 2“Lazaroid” ((−)-2-((4-(2,6-di-1- amino- Pyrrolidinyl-4-pyrimidinyl)-steroid, 1-piperazinyl)methyl)-3,4- Peroxidation dihydro-2,5,7,8-inhibitor tetramethyl-2H-1- benzopyran-6-ol-2HCl) Trolox 0.995 ± 0.019 2Antioxidant (6-Hydroxy-2,5,7,8- tetramethylchroman-2- carboxylic acid)Abbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All ratio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

Supplementary Table 7. Effects of Miscellaneous Compounds (100 μM) onSIRT1 Catalytic Rate. Ratio to Control Rate Mean ± Repli- Compound SEcates Structure & Activities Dipyridamole (2,6-bis- (Diethano- lamino)-4,8-di- piperidino- pyrimido[5,4- d]pyrimidine) 3.54 ±0.20 2

Inhibitor of Adenosine Transport, Phosphodiesterase, 5-LipoxygenaseNicotinamide 0.428 ± 42

0.019 Sirtuin Reaction Product/Inhibitor NF279 0.0035 ± 3

0.0011 Purinergic Receptor Antagonist NF023 −0.0016 ± 3

0.0015 G-protein Antagonist Suramin −0.0002 ± 3

0.0010 G-protein Antagonist, Reverse Transcriptase InhibitorAbbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All ratio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

Supplementary Table 8. Effects of Various Modulators on SIRT1 Rate.Ratio to Compound, Control Rate (Concentration) Mean ± SE ReplicatesStructure ZM 336372, (100 μM) 3.5 ± 1.1 3

Camptothecin, (10 μM) 2.92 ± 0.41 3

Coumestrol, (10 μM) 2.30 ± 0.31 2

NDGA, (100 μM) 1.738 ± 0.088 3

Esculetin, (10 μM) 1.737 ± 0.082 3

Sphingosine 0.069 ± 0.028 3

Abbreviation: SE, Standard error of the mean. Rate measurements with 25μM NAD⁺ and 25 μM p53-382 acetylated peptide substrate were performed asdescribed in Methods. All ratio data were calculated from experiments inwhich the total deacetylation in the control reaction was 0.25-1.25 μMpeptide or 1-5% of the initial concentration of acetylated peptide.

TABLE 9 SIRT1 Rate Effects of New Resveratrol Analogs (100 μM). Ratio toStability in Control Rate Solution Compound Mean ± SE N Structuret_(1/2), hrs. BML-217 (3,5-Dihydroxy- 4′-chloro-trans- stilbene) 10.6 ±0.4  3

Resveratrol (3,5,4′- Trihydroxy-trans- stilbene) 10.4 ± 0.5  43

59 (ethanol), 20 (water) Pinosylvin (3,5-Dihydroxy- trans-stilbene) 9.95± 0.45 3

BML-225 (3,5-Dihydroxy- 4′-ethyl-trans- stilbene) 9.373 ± 0.014 3

BML-212 (3,5-Dihydroxy- 4′-fluoro-trans- stilbene) 8.20 ± 0.69 3

66 (ethanol) BML-228 (3,5-Dihydroxy- 4′-methyl-trans- stilbene) 7.72 ±0.12 3

TABLE 10 SIRT1 Rate Effects of New Resveratrol Analogs (100 μM). Ratioto Stability in Control Rate Solution Compound Mean ± SE N Structuret_(1/2), hrs. BML-232 (3,5-Dihydroxy- 4′-azido-trans- stilbene) 7.24 ±0.12 3

BML-230 (3,5-Dihydroxy- 4′-thiomethyl- trans-stilbene) 6.84 ± 1.26 6

BML-229 (3,5-Dihydroxy- 4′-nitro-trans- stilbene) 6.78 ± 0.22 3

BML-231 (3,5-Dihydroxy- 4′-isopropyl- trans-stilbene) 6.01 ± 0.15 3

BML-233 3,5-Dihydroxy-4′- methoxy-trans- stilbene 5.48 ± 0.33 6

TABLE II SIRT1 Rate Effects of New Resveratrol Analogs (100 μM). Ratioto Stability in Control Rate Solution Compound Mean ± SE N Structuret_(1/2), hrs. Rhapontin aglycone (3,5,3′Trihydroxy- 4′-methoxy-trans-stilbene) 4.060 ± 0.069 3

BML-227 (3,4′-Dihydroxy-5- acetoxy-trans- stilbene) 3.340 ± 0.093 3

BML-221 (3,5-Dihydroxy-4′- acetoxy-trans- stilbene) 3.05 ± 0.54 6

504 (ethanol) BML-218 (E)-1-(3,5- Dihydroxyphenyl)- 2-(2-napthyl) ethene3.05 ± 0.37 6

3-Hydroxystilbene 2.357 ± 0.074 3

TABLE 12 SIRT1 Rate Effects of New Resveratrol Analogs (100 μM). Ratioto Stability in Control Rate Solution Compound Mean ± SE N Structuret_(1/2), hrs. BML-226 (3,5-Dimethoxymethoxy- 4′-thiomethyl-trans-stilbene) 2.316 ± 0.087 3

BML-222 (3,5-Dihydroxy-4′- acetamide-trans- stilbene) 1.88 ± 0.11 3

3,4-Dihydroxy- trans-stilbene 1.64 ± 0.10 6

BML-224 (E)-1-(3,5- Dihydroxyphenyl)- 2-(cyclohexyl) ethene 1.297 ±0.042 3

3,4-Dimethoxy- trans-stilbene 1.127 ± 0.019 3

TABLE 13 SIRT1 Rate Effects of New Resveratrol Analogs (100 μM). Ratioto Stability in Control Rate Solution Compound Mean ± SE N Structuret_(1/2), hrs. Dihydroresveratrol (1-(3,5-Dihydroxyphenyl)-2-(4-hydroxyphenyl) ethane) 1.08 ± 0.14 4

4-Hydroxy-trans- stilbene 0.943 ± 0.039 3

BML-219 N-phenyl-(3,5- dihydroxy)benzamide 0.902 ± 0.014 3

3,5-Dihydroxy-4′- nitro-trans-stilbene 0.870 ± 0.019 3

4-Methoxy-trans-stilbene 0.840 ± 0.089 3

TABLE 14 Resveratrol Analog Synthetic Intermediates CompoundBenzylphosphonate Aldehyde Structure BML-217 (3,5-Dihydroxy-4′-chloro-trans- stilbene) Diethyl 3-5- dimethoxybenzyl phosphonate4-Chlorobenzaldehyde

Resveratrol (3,5,4′- Trihydroxy-trans- stilbene) N/A N/A

Pinosylvin (3,5-Dihydroxy- trans-stilbene) Diethyl benzyl phosphonate3,5-Dimethoxy benzaldehyde

BML-225 (3,5-Dihydroxy- 4′-ethyl-trans- stilbene) Diethyl 3-5-dimethoxybenzyl phosphonate 4-Ethylbenzaldehyde

BML-212 (3,5-Dihydroxy- 4′-fluoro-trans- stilbene) Diethyl 4-fluorobenzylphosphonate 3,5-Dimethoxy benzaldehyde

BML-228 (3,5-Dihydroxy- 4′-methyl-trans- stilbene) Diethyl 3-5-dimethoxybenzyl phosphonate 4-Methylbenzaldehyde

TABLE 15 Resveratrol Analog Synthetic Intermediates CompoundBenzylphosphonate Aldehyde Structure BML-232 (3,5-Dihydroxy-4′-azido-trans- stilbene) Diethyl 4-azido benzylphosphonate3,5-Dimethoxymethoxy benzaldehyde

BML-230 (3,5-Dihydroxy- 4′-thiomethyl- trans-stilbene) Diethyl4-methylthio benzylphosphonate 3,5-Dimethoxymethoxy benzaldehyde

BML-229 (3,5-Dihydroxy- 4-nitro-trans- stilbene) Diethyl 3-5-dimethoxybenzyl phosphonate 4-Nitrobenzaldehyde

BML-231 (3,5-Dihydroxy- 4′-isopropyl- trans-stilbene) Diethyl 3-5-dimethoxybenzyl phosphonate 4-Isopropyl benzaldehyde

3,5-Dihydroxy- 4′-methoxy- trans-stilbene N/A N/A

TABLE 16 Resveratrol Analog Synthetic Intermediates CompoundBenzylphosphonate Aldehyde Structure Rhapontin aglycone(3,5,3′Trihydroxy- 4′-methoxy-trans- stilbene) N/A N/A

BML-227 (3,4′-Dihydroxy-5- acetoxy-trans- stilbene) N/A N/A

BML-221 (3,5-Dihydroxy-4′- acetoxy-trans- stilbene) N/A N/A

BML-218 (E)-1-(3,5- Dihydroxyphenyl)- 2-(2-napthyl) ethene Diethyl 3-5-dimethoxybenzyl phosphonate 2-Naphthaldehyde

BML-216 3-Hydroxystilbene Benzylphosphonate 3-Methoxy benzaldehyde

TABLE 17 Resveratrol Analog Synthetic Intermediates CompoundBenzylphosphonate Aldehyde Structure BML-226 (3,5-Dimethoxymethoxy-4′-thiomethyl- trans-stilbene) Diethyl 4-methylthio benzylphosphonate3,5dimethoxymethoxy benzaldehyde

BML-222 (3,5-Dihydroxy-4′- acetamide-trans- stilbene) Diethyl4-acetamido benzylphosphonate 3,5-dimethoxymethoxy benzaldehyde

BML-215 3,4-Dihydroxy- trans-stilbene Benzylphosphonate 3,4-Dimethoxybenzaldehyde

BML-224 (E)-1-(3,5- Dihydroxyphenyl)- 2-(cyclohexyl) ethene3,5-Dimethoxy benzylphosphonate Cyclohexane carboxaldehyde

3,4-Dimethoxy- trans-stilbene Benzylphosphonate 3,4-Dimethoxybenzaldehyde

TABLE 18 Resveratrol Analog Synthetic Intermediates CompoundBenzylphosphonate Aldehyde Structure Dihydroresveratrol(1-(3,5-Dihydroxyphenyl)- 2-(4-hydroxyphenyl) ethane) N/A N/A

BML-214 4-Hydroxy-trans- stilbene Benzylphosphonate 4-Methoxybenzaldehyde

BML-219 N-phenyl-(3,5- dihydroxy)benzamide N/A N/A

3,5-Dihydroxy-4-nitro- trans-stilbene 3,5-Dimethoxy benzylphosphonate4-Nitrobenzaldehdye

4-Methoxy-trans- stilbene Benzylphosphonate 4-Methoxy benzaldehyde

1. A method for activating a sirtuin deacetylase protein family member,comprising contacting the sirtuin deacetylase protein family member withan activating compound having a formula selected from the groupconsisting of formulas 1-25 and
 30. 2. The method of claim 1, whereinthe compound is a polyphenol compound or analog or derivative thereof.3. The method of claim 1, wherein the plant compound is selected fromthe group consisting of flavones, stilbenes, flavanones, isoflavones,catechins, chalcones, tannins and anthocyanidins or analog or derivativethereof.
 4. The method of claim 1, wherein, if the compound is anaturally occurring compound, it is not in a form in which it isnaturally occurring.
 5. The method of claim 3, wherein the compound isselected from the group consisting of resveratrol, butein, piceatannol,isoliquiritgenin, fisetin, luteolin, 3,6,3′,4′-tetrahydroxyflavone,quercetin, and analogs and derivatives thereof.
 6. The method of claim1, wherein the sirtuin deacetylase protein family member is SIRT1. 7.The method of claim 1, wherein the sirtuin deacetylase protein familymember is in a cell, and the method comprises contacting the cell withthe compound.
 8. The method of claim 7, wherein the cell is in vitro. 9.The method of claim 7, wherein the cell is a cell of a subject.
 10. Themethod of claim 7, wherein the cell is in a subject and the methodcomprises administering the compound to the subject.
 11. The method ofclaim 1, further comprising determining the activity of the sirtuindeacetylase protein family member.
 12. The method of claim 10, furthercomprising determining the activity of the sirtuin deacetylase proteinfamily member.
 13. The method of claim 7, wherein the cell is contactedwith the compound at a concentration of 0.1-100 μM.
 14. The method ofclaim 1, further contacting the cell with an additional activatingcompound having a formula selected from the group consisting of formulas1-25 and
 30. 15. The method of claim 14, comprising contacting the cellwith a least three different activating compounds having a formulaselected from the group consisting of formulas 1-25 and
 30. 16. A methodfor inhibiting the activity of a sirtuin protein family member,comprising contacting the sirtuin deacetylase protein family member withan inhibiting compound having a formula selected from the groupconsisting of formulas 26-29 and
 31. 17. A method for shortening thelifespan of a cell or rendering it resistant to stress, comprisingcontacting the cell with an inhibiting compound having a formulaselected from the group consisting of formulas 26-29 and
 31. 18. Acomposition comprising two compounds each having a formula selected fromthe group of formulas 1-31.
 19. A method for identifying a compound thatmodulates SIRT1 in vivo, comprising (i) contacting a cell comprising aSIRT1 protein with a peptide of p53 comprising an acetylated residue 382in the presence of an inhibitor of class I and class II HDAC underconditions appropriate for SIRT1 to deacetylate the peptide and (ii)determining the level of acetylation of the peptide, wherein a differentlevel of acetylation of the peptide in the presence of the test compoundrelative to the absence of the test compound indicates that the testcompound modulates SIRT1 in vivo.